CA2088665C - Methotrexate derivative - Google Patents

Abstract

Disclosed is a compound represented by the formula:
wherein R1 : CH2, CH2CH2, CH2O, CH2S, CH2S0; R2 ;
hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group; n: an integer of 1 to 4; R3: COOR4, NHCOR5, CONR6R7, PO3H2, SO3H.
The compound shows potent antirheumatic function, psoriasis curing function and carcinostatic function and has low toxicity whereby it is available as a medicine.

Description

~~~~5~u:~~~~
sPECr.F:ccAT:>:orr Novel methotr_~x~t-e deriva~ivg BACKGROUND OF' THE INVENTTON _ 1. Field of the invention This invention relates t o a methot re.xate derivative, more specifically to a novel methotrexate derivative available as an antirheumatic agent, psoriasis curing agent and carcinostatic agent.

2. Prior art Methotrexate has been known as a curing agent of leukemia, and Gubner et al. reported its effectiveness for rheumatoid arthritis (RA) or psoriasis in 1951 and it has been used until then as a curing agent of RA in Europe and the United States. Relatively recently, detailed investigations about method of use and dose have been carried out, and it has been clarified that methotrexate therapy in a low dose develops excellent effectivity with a relatively limited side effects. However, side effects such as hepatopathy or fibroid lung caused by the administration of methotrexate cannot be ignored so that a drug having fewer side effects and increased effectiveness has earnestly been desired.
As methotrexate derivatives to which an alkyl group other than methyl group is introduced at N1~, there have been known for example, the following formula:
1-r2N ' /N N
Et COON
N
CH2N ~ CNHCHCH,2CFI2 COON
ii (J. Med. Chem., 22, p. 862 (1979)) or the formula:
N N
H2N \
Px COOH
N ~ CFIZN ~ ~ - CNHCHCH2CH2 COOH
II
NH2 p - 1 _ (J.Med. Chem. 25, p. 877 (1982)), and the like however, they did not necessarily show sufficient activity.
SUMMARY OF THE INVENTION
The present invention is to provide a novel methotrexate derivative represented by the following formula (I):
T I
H2~~ ~ ~ R
H ~ OOR1 3 (I) ,~ CH2I~ -CNCH(CH2)nR

wherein R' represents CHZ, CH2CH2, CH20, CHZS or CHZSO; R2 represents a 1 o hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group; n represents an integer of 1 to 4; R3 represents a group of formula P03H2, S03H, COOR4 in which R4 represents hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, a group of formula NHCORS in which RS
represents an unsubstituted or substituted phenyl group, or a group of formula CONR6R~ in which R6 represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms; and R' represents a lower alkyl group having I to 4 carbon atoms, an unsubstituted or substituted phenyl group, an unsubstituted or substituted carboxylalkyl group or a lower alkylsulfonyl group.
The compound of the present invention has an excelent antirheumatic 2 0 function, psoriasis curing function and carcinostatic function and is also characterised in having a lower toxicity than that of the convention methotrexate.

BRE;TF DE~~IPTIQN~F~L'IIE DRAWaNC-'~, Figs . 1 to 3 show amounts (ratio) of 3I-1-UdR to be uptake at a respective concentration of drugs to be tested.
Fig. 9 shows "rat keratinocyte growth inhibition-~func-tion" and Fig. 5 shows "human kerat:inocyte growth inhibi-tion function", respectively. Absorbances at respective concentrations of drugs to be tested are shown based on an absorbance when no drug is added as 100 0.
Fig. 6 shows "growth inhibition function of P388 cell"
arid Fig. 7 shows "growth inhibition function of colon 2,(;".
Absorbances at respective concentrations of drugs to be tested are shown based on an absor.bance when no drug is added as 100 0.
Fig. 8 shown weight changes in rats when methotrexate or the compound of the present invention is administered, Fig. 9 shows change in number of white blood cells (GJBC) and red blood cells (RBC), and F9.g. 10 shows changes in liver function and TG content in liver.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The compounds of the present invention are each novel and have not yet been described in any of references and may be synthesized, for example, as follows:
(Method A) Rl 1 I R /
ANN - ~' - COOH ~ AYN~- -COC1 (z>
_R1, C00R2 Condensation ~ ~ COOR2 (2) + 3 ~ A11N ._\ ~~_ CONH ~~(CH2)nk3 H2N " (CH )nR i (3) (tE) ,y a ._ IZ 1.
R
Depro~ection II -CONH (CH2)nR
(S) H2N~N
(5) N
N ~~ CH2X

-rH' H N N

N '~ CH N - - CONH
~~(CH2)nR3 H2 -, Rl H2N ~~ N
N~ ~ + HN --~ COON

IvH2 ($) N \-N ~
CH2N - ~ - COOH

(9) ~ooR2 ~ ~.~ ~.~ ~ ~:! ~3.
c~) _,.
FIZN~~(CHZ)nR'~
(3) Condensation Ii2N\ N N -R1 COOI<2 Iv CH2N - ---~~ CONH ~ (CH2)nR3 (7) (Method C) When R3 in the Formula (I) represents the formula:' COOK' -NHCO
wherein R' represents a hydrogen atom or a lower alkyl group having 1 t o 4 carbon atoms, A2NH (CHZ)nNH2 + /0 (10) 0 (11) COOR2 COOk' Condensaiv.ion A2NH~~ (CH2)nNHCO -(12}

GOOR2 n :i ~/
A2NH~~(CH2)nN ~
\_/
,i (13) CooR2 Q r..
Deprotection.~ ~(CII )nN~~' (14) 0 1 ..
A1N- ~ --COC1 -I- ( I4) (2) RL

COOR2 ii Condensation 7 A1N - ~ CONEL ~ / .
(CH2)nN,\
(15) 0 Deprotection ----j HN CONH ~(CH2)nN
(.16) 0 H2N ~ N
(1G) N

N~I2 (6) H2N N /N~ _ RI
COOR2 d \N ~ CH2N~ .,~~ CONH ~~ (CH2)nN/

li (17) i,J .9 r.~
'~ f-f l ~ ~ ~ fZ T , COOR
COORZ
N
N ~~CFi2N-- ~ - CONHI ~\
' (Cffz)nNfiCO-.
Nfi2 (lf3) wherein Rl, R2, R3 and n have the same meanings as defined above, R' represents hydrogen atom or a lower alkyl group having 1 to 9 carbon atoms, Ax and A? each represents a protective group and X represents a halogen atom.
In Method A, the reaction for obtaining the compound of the formula (2) from the compound of the formula (1) is carried out by suspending the compound of the formula (1) in an acid halogenating agent such as thionyl chloride, oxalyl chloride, etc. and stirring the mixture at room temperature in the co-presence of a catalytic amount of dimethylformamide, etc. In the formulae, as a protective group represented by A1, there may be mentionecl a carbo-benzoxy group, a tosyl group, an acetyl group, etc.
The reaction for obtaining the compound of the formula (4) from the compound of the formula (2) and the compound of the formula (3) is carried out by adding a solution of the compound of the formula (2) dissolved in a solvent such as dichloromethane, etc. to an aqueous solution of the compound of the formula (3) under ice cooling or water cooling and then stirring the mixture at room temperature in the co-presence of an inorganic base such as potassium carbonate, sodium hydroxide, sodium hydrogen carbonate, etc.
The reaction for obtaining the compound of t he formula (5) from the compound of the formula (9) is carried out by adding the compound of the formula (4) to a solution of anisol or phenol, etc. dissolved in a hydrobromide-acetic acid solution and stirring the mixture at ZO °C t o 60 °C, preferably at room temperature. Also, the reaction for obtaining the compound. of the formula (5) from the compound o:P the formula (4) may be carried out by dissolving the compound of the formula (9) in a solvent such as methanol, ethanol, acetic acid, etc, and then stirring the mixture after adding palladium-carbon, under a hydrogen atmosphere at room temperature.
The reaction fox obtaining the compound of the formula (7) from the compound of the formula (6) and the compound of the formula (5) is carried out by reacting the compound of the formula (6) and the compound of the formula (5) in a solvent such as dimethylacetamide, dimet.hylformamide, etc.
at 0 °C to 100 °C, preferably 50 °C to 60 °C under stir-ring. Particularly when R2 is a hydrogen atom, 1N--sodium hydroxide aqueous solution is added t o a solvent such as methanol, ethanol, etc. and stirring the mixture at 0 °C to 60 °C, preferably at 35 °C to obtain the desired compound.
In the formula, as the halogen atom represented by X, there may be mentioned a bromine atom, a chlorine atom, etc.
In Method B, the reaction for obtaining the compound of the formula (9) from the compound of the formula (6) and the compound of the formula (8) is carried out by reacting the compound of the formula (6) and the compound of the formula (8) in a solvent such as dimethylacetamide, dimethylformamide, et c. at 0 °C to 100 °C, preferably 55 °C
under stirring.
The reaction for obtaining the compound of the formula (7) from the compound of the formula (9) and the compound of the formula (3) is carried out by stirring the compound of the formula (9) in the co-presence of diethylphosphoric acid cyanide or dicyclohexylcarbodiimide and 1-hydroxy-benzotriazole, etc. in a solvent such as dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, etc., then adding the compound of the formula (3) and stirring the mixture at 0 °C to 200 °C, preferably 10 °C to 80 °C under stirring.
Particularly when R2 is hydrogen atom, 1N-sodium hydroxide _ g _ aqueous solution is added to tYie mixture and stirrincJ the mixture at 0 °C to GO °C, preaer.ably at morn temperature to obtain the desired compound.
Ln Method C, the reaction for obtaining the compound of the formula (12) from the compound of the formula (10) and the compound of the formula (11) is carried out by dissolving -the compound of the formula (10) in an aprotic solvent such as chloroform, dichloromethane, tetrahydro-furan, dioxane, etc., adding the compound of the formula (11), water and, for example, potassium carbonate, tri-et.hylamine, sodium hydrogen carbonate, pyridine, etc. to the mixture and stirring 'the mixture at room temperature.
In the forrnulae, as a protective group represented by A2, there may be mentioned a carbobenzoxy group, a tosyl group, an acetyl group, etc.
The reaction for obtaining the compound of the formula (13) from the compound of the formula (12) is carried out, in a solvent such as methanol, etc., by stirring at -60 °C
t o -2U °C, preferably -30 °C and, after adding thionyl chloride, refluxing the mixture.
The reaction for obtaining the compound of the formula (14) from the compound of the formula (13) is carried out by dissolving the compound of the formula (13) in ethanol, methanol, tetrahydrofuran, dioxane, etc. and stirring the mixture in the co-presence of palladium-carbon under hydro-gen atmosphere at room temperature. The reaction for obtaining the compound of the formula (15) from the com-pound of the formula (14) and the compound of the formula (2) is carried out by dissolving the compound of 'the formula (2) in dichloromethane, etc., adding the compound of the formula (14) and potassium carbonate or triethyl-amine arid water and stirring the mixture at room tempera-ture, but amidai:ion may be carried out by the mixed acid anhydride method, active ester or active amide method.
The reaction for obtaining the compound of the formula (16) from the compound of the formula (15) is carried aut l~~~i~~''y~s~' by adding hydrobrom:i.de-acetic acid to which ani.sol or phenol is previously dissolved therein to the compound of the formula (:l5) and sliming the mixture at room tempera-lure .
The reaction for obtaining the compound of the formula (17) from the compound of the formula (6) and the compound of the formula (16) is carried out, after stirring in an aprotic solvent such as dimethylacetamide and dimethylform-amide, etc. at 25 °C to 100 °C, preferably 50 °C to 65 °C, stirring the mixture in water containing, for example, triethylamine, potassium carbonate or sodium hydrogen carbonate, etc.
The reaction for obtaining 'the compound (I) from the compound of the formula (17) is carried out in a solvent such as ethanol, etc. by adding an aqueous sodium hydroxide solution and stirring the mixture at room temperature.
The compound represented by the formula (I) obtained according to the present invention has antirheumatic func-tion, psoriasis curing function and carcinostatic function.
Alsa, as compared with methotrexat e, it has less toxicity.
These functions are confirmed by examining the following experiments:
1. Growth inhibition function of human peripheral blood derived lymphocyte (antirheumatic function) 2~ Growth inhibition experiment of rat arid human kera-tinocytes (psoriasis curing function) 3. Mouse cancer cell. growth inhibition experiment (car-cinostatic function) 4, Toxicity comparison of methotrexate (MTX) and the compound of the present invention according to intraperi-toneal continuous administration using rats.
Experimental example 1 Growth inhibition function of human peripheral blood derived lymphocyte (antirheumatic function) (Method) ~~.~~:~i~~Jb~
Lymphocytes were separated from human I:>eripheral blood by using F:icoll-PaqueR and a drug suitably diluted and 105 lymphocytes were cultivated with PHA (0.3 ~.c~/ml.) in 96-hole culture plate for 2 days. Five hours before completion of the cultivation, 3H-UdR (1 ~.Ci/well) was added and uptake of 3H-UdR in lymphocytes was measured by a scintillation counter (here, PHA shows phytohaemagglutinin and UdR
deoxyuridine). Drugs used are as shown below.
Compound 1 H2N"N N _ / \
COOti N
~~ CI(2N CONII ~~~ CUOIi Compound 2 LI2N~ ~/ N
i COON 'v N ( N CH2N ~~~ CONH ~\/V NHCO
Compound 3 H N COOH
__ COON
./
N ~ ~ CH2N--(~ 1 ~- CONH ~~~ NHCO \) Control 1 H2N N N
(MTX) ~ CH3 COOH
I
~~ CH2N T CONH /~COOH

Control 2 H2N~N~ N
Yli N ~' '~ COON
N'J~ CHZN- ~ ~ CONH ~~ COOH

(Results and consideration) In Figs. 1 to 3, ratios based on 3H-UdR uptake of PHA
stimulus lymphocytes adding no drug as 100 % are shown. As clearly seen from Figs. 1 to 3, it was confirmed that the ~~~~iJ 3 compounds of the present invention have excellent growth inhibition function of lymphocytes (antirheumatic function) than those of Control compounds.
Experimental example _2_ Growth inhibition experiment of rat and human kera-tinocytes (psoriasis curing function) (1) Rat keratinocyte growth inhibition experiment 1) The first cultured rat keratinocyte by using Swiss 3T3 cell as a feeder layer was sewed with a concentration of 4 x 104 cells/ml/well to a 24-hole plate treated by Type IV collagen by using MCDB 153/DME/10o FCS medium containing growth factors (insulin, hydrocortisone, EGF, cholera toxin) and cultivated under 5 o C02 and 95 o Air at 37 'C.
2) After 24 hours, it was washed twice at MCDB 153 medium containing no thymidine nor adenine and a medium exchange was carried out with MCDB 153 medium (1.8 mM Ca) containing growth factors (insulin, hydrocortisone, EGF, cholera toxin) and containing no thymidine nor adenine, and after adding a drug thereto, cultivation was carried out under 5 o C02 and 95 % Ai.r at 37 °C for 4 days.
3) 0.5 o MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) was added with 100 ~.1/well and allowing to stand at 37 °C for 4 hours, and the medium was sucked up. Dye (formazan) formed by living cells was dis-solved by adding DMSO (dimethylsulfoxide) with 200 ~.l/well.
Absorbance (A 540) of the dye dissolved was measured and an average of 6-hole was calculated as a relative value of number of cells.
(2) Human keratinocyte growth inhibition experiment 1) Human keratinocyte obtained from Kurabou K.K. was sewed with a concentration of 2 x 104 cells/ml/well to a 24-hole plate treated by Type TV collagen by using MCDB 153 medium (0.15 mM Ca) containing growth factors (insulin, hydrocortisone, rhEGF, ethanolamine, phosphoethanolamine) and containing no thymidine nor adenine, and cultivated under 5 o C02 and 95 o Air at 37 °C.

~~~r~~~
2) After. 2~1 hours, a specimen ways added thereto and the mixture was cultivated under 5 % C02 and 95 % Air. at 37 °C for 4 days.
3) 0.5 o MTT (3-(~1,5-dimethylthiazol-2-yl)-2,5-di-phenylt etrazolium bromide) was added with 100 ~.1/well. and allowing to stand at 37 °C for 4 hours, and the medium was sucked up. Dye (formazan) formed by living cells was dis-solved by adding DMSO (dimethylsulfoxide) with 200 ~.1/well.
Absorbance (A 550) of the dye dissolved was measured and an average of 6-hole was calculated as a relative value of number of cells.
Drugs used are as shown be7.ow. .
Compound 1 HZN ~.N N
COON
\~N ~CH2N -,~ '~-CONH ~ COOI-I
NH2 ~--J
Compound 3 H2N' JN ~N ~ \ OH

N /~ ~ NHCO -~~
CH2N'~ ~~'v - CONH ~ -Control 1 H2N ~ N ~ N
(MTX) ~ 1 CH COOH
3 /~
N CH2N - ~ -CONH~n CUOH
I

(Results and consideration) In Fig. 4, the results of "rat keratinocyte growth inhibition experiment" are shown and in Fig. 5, the results of "human keratinocyte growth inhibition experiment" are shown. Each is shown as a ratio based on the absorbance to which no drug is added as 100 0.
As clearly seen from Fig. 4 and Fig. 5, it was con-firmed that the compounds of the present invention have ''t:~ ~ u~ ~ ~i excellent kerati.nocyte cJ:rowth inhi,biti.on function (psoria-sis curing function) than that of Control compound.
Ex~oe.rim~nt~l _,ex~m_pl~~
Mouse cancer cell growth inhibition experiment (car-cinostatic function) (Method) Mouse lymphoid neoplasma P388 cell floated by RPM
11640 medium (added so as to have 5 % of bovine fetal serum and 10-6 M of 2-mercaptoethanol), or mouse colon 26 carcinoma cell were placed in each well of a flat-bottom microplate (available from Corning Co., 1k25870) with 5 x 103, and a drug solution prepared in the same medium was placed so as to have 0.2 ml of the drug in each well.
These were cultivated in a carbonic gas cultivating device (carbonic gas 5 0, air 95 %, humidity 100 %) for 3 days and then MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide was dissolved in phosphate buffer physiological saline solution (pH 7.4, 4.10 mM) with an amount of 5 mg/ml) was added to each well with an amount of ~,1, and cultivated for further 4 hours. Cultivation supernatant was removed each 150 ~,1 and formed dye (formazan) was dissolved in DMSO (dimethylsulfoxide; 150 ~.1/well), and absorbance (540 nm) was measured by a spectrophotometer for microplate.
Drugs used are as shown below.
Compound 1 H2N , -COON
N
N CH2N-'~--CONr~~ COON

Compound 3 H N ~ OH
2 ~'~~/ ~ '--COON
y E r--~.,, J
N . CH2N--(,~ ,~~--CONH ~~~ NHCO
NH2 ~~; ~ .
Control 1 11~~5~~~i~i<
tl~N , j, rl Ctl COON
(MTX) 3 ~,~___ N ~'~ CHZN ~~! , \'- CONH ~\ COOtI

(Results and consideration) In Fig. 6, example o.f us:ing P388 cell was shown and in Fig. 7, example of using colon 26 was shown. The data show with %, by cell growth at a well adding no drug, i.e. its absorbance as 100 %, and the degree of inhibiting growth by addition of drugs.
As clearly seen from fi.g. 6 and Fig. 7, it was con-firmed that the compounds of the present invention have a far better cancer cell growth inhibition function (carcino-static function) than that of Control drug.
Experiment ~l example 9 Toxicity comparison of methotrexate (MTX) and the compound of the present invention according to intraperi-toneal continuous administration using rats (Method) To 8-week old SD series male rat was abdominary administered MTX or 'the compound of the present invention one per day with a ratio of 5 days per week for 5 weeks continuously. Doses administered are two degrees of 0.25 and 0.5 mg/kg in each of MTX and the compound of the present invention. For control group, a solvent (phosphate buffer, pH 7.4) was administered similarly. Number of rats constituting one group are five.
Inspected items are as follows.
1) Alive or dead and symptoms: observed every day.
2) Change in body weight: measured once per week.
3) Blood inspection: at final week for administration, numbers of white blood cells (WBC) and red blood cells (RBC) were measured.
4) Liver weight and triglyceride (TG) content in liver:
after final administration, anatomy was effected and liver weight and TG content in liver were measured.

i ) ~~
Drugs used are as shown below.
Compound 3 IiZN ~~~ COOH
COUH
t \ . ~ ~ J~~~/ NHCO~_ cH2N .~-~-corlH
LIZ
Control 1 H?N\ /N ~
~i CI-I~ COOH
(MTX) I
N~~~/ CHZN ~~~>-- CONH ~,~~~ C00H
NHZ
(Results) 1) Alive or dead and symptoms Among MTX administered groups, in a group administered with a dose of 0.25 mg/kg, n.o mouse died and abnormal symptoms were not observed. However, in a group administered with a dose of 0.5 mg/kg, bad nutriture, anemia, decrease in amount of dejection, swelling at mouse peripheral, etc. were observed and one o.f the five died.
On the other hand, in the group in which the compound of the present invention was administered, no mouse died in either 0.25 mglkg group or 0.5 mg/kg group and no abnormal symptoms were observed.
2) Change in body weight In Fig. 8, change in body weight is shown. Prohibi-tion in body weight increment was weak in the compound of the present invention than that of MTX.
3) Blood inspection In Fig. 9, the results of blood inspection are shown.
Decrease in wBC and RBC are clearly weak in the compound of the present invention than that of MTX.
9) Liver weight and TG content in liver In Fig. 10, liver weight and TG content in liver are shown. Increment in liver weight and TG content in liver are both lower with the compound of the present invention than those of MTX.

~~~~~~a (Consideration) Toxicit:i.es of MTX and the compound of the present invention were compared by using rats due to abdornina.l continuous administration. As the results, in all of the points of the death ratio, symptom, body weight change, blood inspection, liver weight and TG content in liver, it can be considered to be low in toxicity of the compound of the present invent:i.on than those of MTX.
EXAMPLES
Reference example :L
S~n~hesis of 1-carbobenzo~-5-carboxyindolin~
To a mixture of water (20 ml) and diethyl ether (20 ml) were added 5-carboxyindoline (2.0 g) and sodium hydr-oxide (0.6 g), and then, under ice-water cooling, carbo-benzoxy chloride (2.59 g) and water (10 ml) containing sodium hydroxide (2.1 g) were added to the mixture thereto alternatively. The mixture was stirred at room temperature for 2 hours. The reaction mixture was made acidic with 2N-hydrochloric acid and precipitated crystals were collected by filtration. Precipitates were washed with ether and air-dried to obtain the title compound (2.8 g).
1H-NMR (DMSO-d6, b) : 3.10 (2H, t, J=8F-Iz) , 4 .03 (2H, t, J=8Hz), 5.23 (2H, s), 7.2-8.0 (8H, m).
mp; 1y4-:Lyo -C:
Reference example 2 Synthesis of diethyl N-(1-carboxybenzox~indolin-5-~arbonyl)-L-glutamate After suspending the compound (2.5 g) of Reference example 1 in thionyl chloride (10 ml), a catalytic amount of dimethylformamide was added to the mixture and the mix-ture was stirred at room temperature for 30 minutes. Then, excessive thionyl chloride was removed under reduced pres-sure and the residue was t.riturated by n-hexane. The resulting crystals were filtered and dissolved in dichloro-methane (20 ml), and the dichloromethane solution was added dropwise t o a water (50 ml) suspension containing diethyl glutamate hydrochloride (3.0 g) and t.riethy:Larnine (2.8 g) under ice--water cooling. After stirring the mixture at room temperature for 2.5 hours, the solvent was removed under reduced pressure and to the residue was added a mixed solution of ethyl acetate (200 ml) and dil. hydrochloric acid (200 ml) under ice-water cooling. After stirring the mixture for 5 minutes, the organic layer was obtained by separation. The organic layer was washed with 5 o aqueous sodium hydrogen carbonate solution and then dried over magnesium sulfate. Then, ethyl acetate was rernoved under reduced pressure, and the residue was subjected to silica gel column chromatography using a mixed solvent of chloroform : methanol = 30 . 1 as an eluent to obtain the title compound (3.1 g).
1H-NMR (CDC13, b): 1.19 (3H, t, J=7Hz), 1.25 (3H, t, J=7I-Iz) , 2.1-2 . 6 (4H, m) , 3 .06 (211, t, J=8Hz), 3.8-4.3 (6.H, m) 4.75 (1H, m), 5.20 (2H, s) , 6.79 (1H, d, J=7I-lz) , 7.2-7 . 7 ( 8H, m) .
mp; 120-121 °C
Reference example 3 Synthesis of diethyl N-(indolin-5-carbon~rl)-L-alutamate The compound (1.8 g) of Reference example 2 was dissolved in tetrahydrofuran (80 ml), and after adding 10 0 palladium-carbon (0.4 g), the mixture was stirred under hydrogen atmosphere for 5 hours. Palladium-carbon was removed by filtration using Celite to obtain the title compound (1.2 g) 1H-NMR (CDC13, 8): 1.21 (3H, t, J=7Hz), 1.29 (3H, t, J=7Hz) , 2.0-2 .6 (4H, m) , 3 . 00 (2H, t, J=8Hz), 3.59 (2H, t, J=8Hz), 4.07 (2H, q, J=7Hz ) , 4 . 19 (2H, q, J=7I-lz ) 9 . 75 ( 1H, m) , 6. 47 (1H, d, J=9Hz) , 6 . 68 (11-I, d, J=7Hz), 7.45 (1H, d, J=9Hz), 7.49 (1H, ~~~~Sy~~i mp; 96-97 °C
Example 1 ~y_zZthe.sis of digif,~yl 1'~1_.-f l2, li.am:LnQ-6- ri inyl),-, metl~,yl l indolin-5-~ar~onyl l -L-cllutamate_ In dimethylacetamide (3 ml) were suspended the com-pound (214 mg) of Reference example 3 and 6-bromomethyl-2,4-diaminopteridine hydrobromate~isopropanol adduct (250 mg), and the mixture was stirred at 50-55 °C for 4 hours.
After cooling, water (15 ml) containing triethylamine (124 mg) was added to the .reaction mixture, and then the mixture was extracted with chloroform (350 m1) dividing into four times. After the organic layer was dried over magnesium sulfate, the solvent was removed under reduced pressure and the residue subjected to silica gel column chromatography using a mixed solvent of chloroform : met hanol = 10 . 1 as an eluent to obtain the title compound (200 mg).
1H-NMR (DMSO-d6, 8) : 1 .22 (3H, t, J=7Hz) , 1 .30 (3H, t, J=7Hz), 2.0-2.5 (4H, m), 3.07 (2H, t, J=8Hz), 3.57 (2H, t, J=8Hz), 4.10 (2H, q, J=7Hz), 4.23 (2H, q, J=7Hz) 4.79 (1H, m) , 5.24 (2H, s) , 6.51 (1H, d, J= 9Hz) , 6.76 (1H, d, J=7Hz) , 7 .57 (1H, s) , 7.59 (1H, d, J=9Hz) , 8.82 (1H, s) .
mp: 168-170 °C
Example 2 synthesis of N-f1-f(2,,4-diamino-6-pteridinyl?methyll-in lin-5-carbonyl(-L-glutamic acid (Compound 1) The compound (170 mg) of Example 1 was dissolved in ethanol (33 ml), and 1N aqueous sodium hydroxide solution (0.84 ml) was added to the solution at 35 °C and the mix-ture was stirred at the same temperature for 9.5 hours.
After continuing stirring at 25 °C .for further 20 hours, water (2 ml) was added to the reaction mixture. The reac-tion mixture was evaporated to dryness under reduced pres-sure. The residue obtained was dissolved in water (15 ml), ~~~Se'Scif~.
and the solut ion wa s adjusted to pf-1=3.7 with 1D1-hydrachlor-ic acid under i.ce-water cooling and allowed to stand at cool place overni-ght. Deposited precipitates were collected by filtration to obtain the title compound (130 mg) .
1H-NMR (DMSO-d6, 8) : 1 . 94 (2F-1, m) , 2 . 32 (21-1, m) , 2 . 98 (2H, t, J=81-lz) , 3.56 (2H, t, J=8Hz) , 4.29 (1H, m) , 4 .53 (2H, s) , 6.71 (1H, d, J=9Hz) , 7 .57 (1F-1, s) , 7.59 (1H, d, J=9Hz) , 8.72 (113, s) .
mp; 201-204 °C (decomposed) Reference example 4 ~ynthe~is of dime~hyl N-(1-carbobenzo~indolin-S-carbonylL-L-a-aminoadipate After suspending 1-carbobenzoxyindolin-5-carboxylic acid (3.1 g) in th.ionyl chloride (10 ml), a catalytic amount of dimethylformamide was added to the suspension and the mixture was stirred at room temperature for 2 hours.
Then, excessive thionyl chloride was removed under reduced pressure and the residue was t riturated by n-hexane. The resulting crystals were filtered and dissolved in dichloro-methane (30 ml), a.nd the dichloromethane solution was added dropwise to an aqueous solution (30 ml) containing dimethyl L-a-aminoadipate hydrochloride (2.7 g) under ice-water cooling. To the reaction solution was further added potas-sium carbonate (5.6 g). After stirring the mixture at room temperature overnight, the reaction mixture was poured into a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The chloroform layer was washed with 1N-hydrochloric acid and dried over sodium sulfate, and then the solvent was removed under reduced pressure.
Then, the residue obtained was subjected to silica gel column chromatography using a mixed solvent of chloroform methanol = 100 . 1 as an eluent to obtain the title compound (3.1 g).
1H-NMR (CDC13, 8) : 1 . 6-2. 1 (4H, m) , 2. 36 (2H, t, J=6. 8Hz) , ~~ I(~~.~' 3.:1.3 (2I-t, rn) , 3.66 (3H, s) , 3.77 (3H, s) , 4 . 09 (2I-I, m) , 4 .78 (1I-I, m) , 5 .27 (2FI, bs), 6.80 (1FI, d, J=7.8I-Iz), 7.2-7..'>
(6H, m) , 7. 63 (2FI, m) .
R~.fer. ncP example synthesis o.f dimethxl N~inc~glin=~~~rbQ,nyl)-L-a-aminopdipate To a 30 o hydrogen bromide-acetic acid (15 ml) solu-tion containing anisole (1.5 g) was added the compound (1.5 g) of Reference example 4 and the m:i.xture was stirred at room temperature for 4 hours. Then, when a large amount of ether was added to the reaction mixture, a red--brownish oily product was precipitated. Almost all the ether layer was removed and the oily product was suspended in chloroform. The suspension was washed with a saturated aqueous sodium hydrogen carbonate solution and the chloro-form layer was collected by separation. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure to obtain the tit7.e compound (g60 mg).
zH-NMR (CDC13, b): 1.6-2.1 (4H, m), 2.36 (2H, t, J=6.8Hz), 3.07 (2H, m) , 3. 66 (3H, s) , 3. 66 (2H, m) 3.77 (2H, s), 4.78 (1H, m), 6.62 (2H, m) , 7 .54 (2H, m) .
Example 3 Synthesis of dimeth~l N-f1-f(2,4-diamino-6-pteridin-yl)methyll-indolin-5-carbonyll-L-a-aminoadipat~
In dimethylacetamide (20 ml) were suspended the compound (960 mg) of Reference example 5 and 6-bromomethyl-2,4-diaminopteridine hydrobromide~isopropanol adduct (1140 mg) and the suspension was stirred at 50 to 60 °C for 6 hours. After cooling, the mixture was poured into a satu-rated aqueous sodium hydrogen carbonate solution, and the desired product was extracted with chloroform dividing into three times. The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure.

''~~~3~y~i~r'~
The .residue obtained was applied to silica gel column chromatography by using a mixed solvent of chloroform methanol = 100 : 10 as an eluent to obtain the title compound (520 mg).
1H-NMR (CDC13, 8) : 1 . 6-2. 1 (4H, m) , 2. 38 (2I-I, 't, J=6. 8Hz) , 3.07 (2i-t, m) , 3.57 (2I-I, m) , 3. 67 (3H, s) 3.78 (3H, s) , 4 .53 (2H, s) , 4 .74 (1H, m) , 6.52 (1I-I, d, J=8.3I-Iz) , 7 . 01 (1H, d, J=7.8Hz), 7.57 (2H, m), 8.77 (1H, s).
Example 4 ~nthesis of N- f 1- f (2 9-diamino-6-~teridinxl ) rnP~y_l.~-indolin-~-c~rt~on,~l l -L-a-aminoadigic amid The compound (400 mg) of Example 3 was dissolved in ethanol (22 ml), and 1N aqueous sodium hydroxide solution (3.1 ml) was added to the solution at 35 °C and the mixture was stirred at the same temperature for 4 hours. After continuing stirring at 25 °C for further 20 hours, water (3 ml) was added to the reaction mixture, and the reaction mixture was evaporated t o dryness under reduced pressure.
During this procedure, the outer temperature was controlled so as not to exceed 30 °C. A yellowish solid material was obtained dissolved in water (10 ml), and the solution was adjusted to pH=3.7 with 1N-hydrochloric acid and allowed to stand in a refrigerator for 2 hours. Deposited precipitates were collected by filtration to obtain the title compound (320 mg).
1H-NMR (CDC13, 8): 1.4-1.9 (4H, m), 2.23 (2H, t, J=6.8Hz), 3. O1 (2H, m) , 3.58 (2H, m) , 4 .32 (1H, m), 4.55 (2H, s), 6.69 (1H, d, J=8.3Hz), 7 . 63 (2H, m) , 8.1.0 (1H, d, J=8 .3Hz) , 8.72. (1H, s) .
Reference example 6 nthesis of Na-(1-carbobenzoxy_indolin-5-carbonyl)-NS-benzoyl-L-ornithine me~t~l ester To thionyl chloride (1.5 ml) was added 1-carbobenzoxy-indolin-5-carboxylic acid (180 mg) to make a suspension, t~~~~~'raG3 and a catalytic amount of dimethyl:Eormarn.i.de was added to the suspension and the mixture was stirred at roorn tempera-ture far 2 hours. Next, the mixture was evaporated to dry-ness under reduced pressure. The solid material obtained was dissolved in dichloromethane (4 rnl), and to the solu-tion were added Ns-benzoyl-L-ornithine methyl ester (150 mg) and potassium carbonate (750 mg), and water (4 ml) was further added thereto and the mixture vigorously stirred at room temperature for 12 hours. Then, the reaction mixture was poured into water, extracted with chloroform and the chloroform layer was washed with a 1N-hydrochloric acid solution and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue obtained was applied to silica gel chromatography by using chloroform methanol = 100 . 3 as an eluent to obtain the title com-pound (140 mg) .
~H-NMR (CDC13, b) : 1 . 6-2.2 (4H, m) , 3. 15 (2H, t, J=8.8I-Iz) , 3.56 (2H, m) , 3.78 (3H, s) , 4 .10 (2H, t, J=8.8Hz), 4.82 (1H, m), 5.27 (2H, s), 6.70 (1H, m), 6.89 (1H, d, J=8.8Hz), 7.20 (8H, m), 7.80 (2H, m) . .
Reference example 7 Synthesis-of Na-(indolin-5-carbonyl?-NS-benzo~l-L-ornithine methyl ester The compound (140 mg) of Reference example 6 was added to a 30 o hydrogen bromide-acetic acid (2 ml) of phenol (150 mg), and the mixture was stirred at roorn temperature for 4 hours. Next, when a large amount of ether was added to the reaction solution, red-brownish oily product was precipitated. Almost all the part of the ether layer was removed and the oily product was suspended in chloroform.
The suspension was washed with a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform.
The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure to obtain the title compound (50 mg).

lI-1-NMR (CDC13, 8) : 1 . 6-2.1 (4H, m) , 3.02 (2Ht, t, J=8.8Hz) , 3.56 (2I-I, m) , 3. 6?. (2H, t, J=8 . 8Hz) , 3.75 (3I-I, s) , 4.79 (1H, rn) , 6.55 (1H, d, J=7 . 8Hz) , 6. 86 (2H, m) , '6. 99 (1F-I, rn) , 7 . 1-7 . 6 (5H, m) , 7 . 82 (2H, , m) .
Example 5 ~~rnthesis of Na~1- f (2, 4-~' amino-6-pteridinyl) me~:hyl l -indolin-5-car~~ny1)-Ns-benzo~l-L-arnithin~ meth~rl ester_ In dimethylacetamide (1 ml) were suspended the com-pound (50 mg) of Reference example 7 and 6-bromomethyl--2,4-diaminopteridine hydrobromide~isopropanol adduct (44 rng), and the mixture was stirred at 50 to 55 °C for 4 hours.
After cooling, water (3 ml) containing t riethylamine (22 mg) was added to the reaction mixture, and 'then the mixture was extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was removed under reduced pressure and the residue was applied to silica gel chromatography using a mixed solvent of chloro-form : methanol = 10 : 1 as an eluent to obtain the title compound (34 mg).
1H-NMR (CDC13, 8) : 1 . 6-2 .2 (4H, m) , 3. 07 (2H, t, J=8 . SHz) , 3 .50 (2H, m) , 3 .56 (2f-I, t, J=8 . 8I-Iz) , 3.79 (3H, s), 4.53 (2H, s), 4.76 (1H, s), 6.52 (1H, d, J=8.3Hz), 7.19 (1H, d, J=7.6Hz), 7.45 (3H, m), 7.62 (2H, m), 7.80 (2f-I, m), 8.76 (1H, s) .
Example 6 Synthesis of Na-f1-f(2,4-diamino-6-pteridinyl)methyll-indolin-5-carbonyl)-Ns-benzoyl-L-arnithine In ethanol (5 ml) was dissolved the compound (34 mg) of Example 5, and 1N-sodium hydroxide aqueous solution (0.1 ml) was further added to the solution and the mixture was stirred at 35 °C for 4.5 hours. After stirring at 25 °C
for 20 hours, water (1 ml) was added to the reaction mix-ture and the reaction mixture was evaporated to dryness under reduced pressure. During this procedure, the outer temperature was controlled so as not to exr_eed 30 °C:. A
yellowish solid material. was obtained and dissolved in water (5 ml), and the solution was adjusted to pI-I=3.7 with 1N-hydrochloric acid and allowed to stand in a refrigerator for 2 hours. Deposited precipitates were collected by filtration to obtain the title compound (26 mg).
lI-I-NMR (DMSO-d6, 8) : 1 . 64 (2H, m) , 7.. 83 (2H, m) , 2. 98 (2H, t, J=8.3Hz), 3.57 (2H, t, J=8.3Hz), 4 .36 (1FI, m) , 4.53 (2H, s) , 6. 64 (2H, m) , 7 .45 (3I-I, m) , 7 .59 (2H, m) , 7 . 82 (2H, m) , 8 . 12 (1H, d, J=8 . 6Hz) , 8 . 43 (1I3, m), 8.69 (1H, s) .
mp; 179-183 °C (decomposed) Reference example Synthesis of Ns-phthalo~l-Na-carbobenz2xy-ornithine methyl ester Phthalic anhydride (2.45 g) was added to a dichloro-methane (70 ml) solution of Na-carbobenzoxy-L-ornithine (2.0 g), and then water (70 ml) and potassium carbonate (1.12 g) were added to the mixture and the mixture was stirred at room temperature for 15 hours. The mixture was condensed to 60 ml under reduced pressure and adjusted to pH=3 with 1N-hydrochloric acid. Deposited precipitates were collected by filtrat9.on and vacuum dried. White crystals obtained were dissolved in low water-content methanol (80 ml) and the solution was cooled to -30 °C and stirred for 10 minutes. Then, at the same temperature, thionyl chloride (2 ml) was slowly added dropwise. The reaction mixture was slowly returned to room temperature and refluxed for a further 2 hours. The solvent was removed under reduced pressure and the residue obtained was applied to silica gel chrornatography by using chloroform methanol = 100 . 1 as an eluent to obtain the title compound (2.16 g).
1H-NMR (CDC13, 8): 1.6-2.0 (4H, m), 3.69 (5H, m), 4.20 (1H, m), 5.06 (2H, s), 5.70 (1H, m), 7.29 A
(5f1, rn) , 7 . 66 (2I-1, rn) , 7 . 81 (2H, rn) .
Reference example.
SynthPSi.s of Ns-phthaloyl.- rn:i .h:i.ne methy:L Ps r After adding a 10 o palladium-carbon (500 mg) to a methanol solution (100 ml) of the compound (2.16 g) of Reference example 8, the mixture was stirred under hydrogen atmosphere at room temperature fo.r 20 hours. Palladium-carbon was removed by filtration using Celite and the sol-vent was removed under reduced pressure. The residue obtained was subjected to silica gel chrornatography by using chloroform : methanol = 100 . 3 as an eluent to obtain the title compound (223 mg).
1H-NMR (CDCIg, b) : 1 . 7-2.1 (4I-1, m) , 3. 75 (2r1, m) , 3.83 (3H, s) , '7 .76 (2H, m) , 7 . 84 (2.H, m) .
Reference example 1.0 Synthesis of Na-(1-carbobenzox ~indoli.n-5-carbonyl)-NS_ phthaloyl-ornithine methyl ester By adding thionyl chloride (2.5 ml) to 1-carbobenzoxy-indolin-5-carboxylic acid (297 mg) to prepare suspension, and a catalytic amount of dimethylformamide was added to the suspension and the mixture was stirred at room temperature for 2 hours. Then, the mixture was evaporated to dryness under reduced pressure. The resulting solid material was dissolved in dichloromethane (7 ml), and to the solution were added the compound (250 mg) of Reference example 9 and potassium carbonate (640 mg). Then, water (7 ml) was further added thereto, and 'the mixture was vigorously stirred at room temperature for 12 hours. Next, the reaction mixture was poured into water and extracted with chloroform, and the chloroform layer was washed with 1N-hydrochloric acid and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography using chloroform : methanol = 100 . 3 as an eluent to obtain the title compound (330 mg).
1H-NMR (CDClg, 8): 1.6-2.1 (9H, m), 3.12 (2H, t, J~8.8Hz), 3. 'J2 (2ff, m) , 3.'76 (3Ff, s) , 4.08 (2f-1, t, J=8.8f-Iz) , 4 .84 (1H, m) , 5.2 7 (2H, s) , 6. 80 ( 1H, d, J=7 . BEIz) , '7 . 1-7 .5 ( 6If, m) , 7 . 5-7 . 9 ( 6H, m) .
Reference example 11 Synth_~sis of Na~indolin-5-car_bonv_1_LNS-phthaloyl--grnithi.ne methyl ester To a 30 o hydrogen bromide-acetic acid (8 ml) solution of phenol (300 mg) was added the compound (330 mg) of Reference example 10 and the mixture was stirred at room temperature for 4 hours. Then, when a large amount of ether was added to the reaction mixture, a red-brownish oily product was precipitated. Almost all the ether layer was removed and 'the oily product was suspended in chloroform. The suspension was washed with a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure to obtain the title compound (147 mg).
1H-NMR (CDC13, b) : 1 . 6-2 .1 (4H, m) , 3. 04 (2H, t, J=8. 3Hz) , 3.62 (2H, t, J=8.3Hz), 3.72 (2H, m), 3.75 (3H, s), 4.86 (1H, m), 6.5-6.7 (2H, m), 7.52 (2H, m), 7.68 (2H, m), 7.82 (2H, m) .
Example 7 Synthesis of Na-f1-ff2,4-diamino-6-pteridinyl)methyll-indolin-5-carbonyl) -NS phthaloyl-ornithine methyl ester In dimethylacetamide (1.0 ml) were suspended the com-pound (146 mg) of Reference example 11 and 6-bromomethyl-2,4-diaminopteridine hydrobromide~isopropanol adduct (115 mg) and the suspension was stirred at 50 t o 55 °C for 9 hours. After cooling, to the mixture was added water (4 ml) containing triethylamine (30 mg), and the mixture was stirred and then extracted with chloroform. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue obtained was p i ~~.~t~~~~lPj applied to si.l.ica gel chrarnatography by using a mixed solvent of chloroform : methanal = 1.0 . 1 as an eluent to obtain the title compound (140 mg), 1H-NMR (CDC13, b) : 1.7-2.2 (4EI, m), 3.06 (2H, t, J---8.3Hz), 3.56 (2H, t, J=8.3Hz), 3.72 (2H, m), 3.76 (3H, s) , 4._'i3 (2I-I, s) , 4.88 (1H, m), 6.45-6.62 (2H, m), 7.56 (2H, m), 7.71 (2H, m) , 7 . 84 (2H, m) , 8. 82 (1H, s) .
example 8 Synthesis of Na-f1-f12,4-diami:no-6-pteridin~t)methyll indolin-5-c~rbonvl)-NS--h~miphthalc~y:L-o_rni~hine (Compound~i, The compound (140 mg) of Example 7 was suspended in 2N-sodium hydroxide aqueous solution (10 ml), and the mixture was stirred at 30 °C for 12 hours. The mixture was evaporated to dryness under .reduced pressure and yellowish solid material obtained was dissolved in water (5 ml). The solution was adjusted to pH=3.7 with 1N-hydrochloric acid and allowed to stand in a refrigerator for 2 hours. Depo-sited precipitates were collected by filtration. A
yellowish solid material was obtained subjected to silica gel chromatography by using a mixed solvent of chloroform methanol: 28 o-aqueous ammonia = 5 : 4 . 1 as an eluent to obtain the title compound (20 mg).
1H-NMR (DMSO-d6, 8): 1.5-2.1 (4H, m), 3.14 (2H, t, J=
8.3Hz) , 3.58 (2I-I, t, J=8.3Hz) , 4.38 (1I3, m) , 4 .54 (2I3, s) , 6.71 (1H, m) , 7.3-7.6 (4H, m), 7.6-7.8 (3H, m), 8.08 (1H, m) , 8.71 (1H, s) .
mp; 195-199 °C (decomposed) Reference example 12 Synthesis o:E N&-(3-methoxycarbonylbenzoyl)Na-carbo-benzoxy-ornithine methyl ester To a dichloromethane (40 ml) solution of Na-carbo-benzoxy-L-ornithine (2.9 g) was added methyl isophthalate chloride (2.1 g), and then, water (40 ml) and potassium ~,~~~9i~~~~q3 carbonate (2.4 c~) we.re added to the mixture a.nd the rni.xt.ure was stirred at room temperature for. 15 hours. fhe rnixture was condensed under reduced pressure to 30 ml and adjusted to pH=3 with 1N-hydroch.lori.c acid, and deposited precipi-fates were collected by filtration and vacuum dried. A
white solid was obtained and dissolved in a low water-content methanol (100 ml) and the solution was cooled to -30 °C and stirred .for 10 minutes. Then, thionyl. chloride (3 ml) was gradually added dropwise at the same temperature. The reaction solution was slowly returned to room temperature and refluxed for a further 2 hours. The solvent was removed under reduced pressure and the residue obtained was subjected to silica gel chromatography by using chloroform : methanol = 100 . 1 as an eluent to obtain the title compound (600 mg).
1H-NMR (CDC13, 8): :L.6-2.0 (4H, m), 3.46 (2H, m), 3.70 (3H, s), 3.89 (3H, s), 4.34 (1H, m), 5.08 (2H, s), 5.88 (1H, d, J=7.8Hz), 7.31 (5H, s), 7.45 (1H, m), 8.05 (2H, m), 8.41 (1H, s) .
Reference example 13 Synthesis of Nb- (3-methoxycarbon~lbenz ~lZ-ornithin~, methyl ester After adding a 10 % palladium-carbon (100 mg) to a methanol solution (100 ml) of 'the compound (600 mg) of Reference example 12, the mixture was stirred under a hydrogen atmosphere at room temperature for 20 hours.
Palladium-carbon was removed by filtration using Celite and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography by using chloroform : methanol = 100 . 3 as an eluent to obtain the title compound (360 mg).
1H-NMR (CDC13, 8) : 1 . 6-2 . 0 (4H, m) , 3. 50 (3H, m) , 3 .72 (3H, s), 3.92 (3H, s), 7.99 (1H, t, J=7.8Hz), 7.63 (1H, m), $.0-8.2 (2H, m), 8.43 (1H, s) .

Reference example 14, ~r~.h'~Sis o~ N_a (1-carbobenz x i. Olin =~~:arbonyl) -NS_ (3-methoxy~arbonylbenz_~~rnit.h_in rc~athy~ ester By adding thionyl chloride (5 ml) to 1-carbobenzoxy-indolin-5-carboxylic acid (350 mg) to prepare suspension, and a catalytic amount of dimethylf:ormamide was further added to the suspension and the mixture was stirred at room temperature for 2 hours. Then, the mixture was evaporated to dryness under reduced pressure. ThP .resulting solid material was dissolved in dichloromethane (7 ml), and to the solution were added the compound (360 mg) of Reference example 13 and potassium carbonate (650 mrJ). Then, water (7 ml) was further added thereto, and the mixture was vigorously stirred at room temperature for 12 hours. Next, the reaction mixture was poured into water and extracted with chloroform, and the chloroform layer was washed with 1N-hydrochloric acid and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography using chloroform : methanol = 100 . 3 as an eluent to obtain the title compound (390 mg) .
1H-NMR (CDC13, 8): 1.6-2.1 (9H, m), 3.08 (2H, t, J=8.3Hz), 3.52' (2H, m) , 3.75 (3H, s) , 3. 88 (3H, s), 4.06 (2H, t, J=8.3Hz), 4.78 (1H, m), 5.26 (2I3, bs) , 7 .16 (1H, d, J=7 . 3I-Iz) , 7.2-7.6 (7H, m), 7.65 (2H, m), 7.9-8.1 (2H, m) , 8.93 (1H, s) .
Reference example 15 Synthesis of Na- (indo)_in-5-carbonylZ Ns- (3-methoxy-carbonylbenzoyl)-ornithine methyl ester To a 30 o hydrogen bromide-acetic acid (6 ml) solution of anisol (0.5 g) was added the compound (390 rng) of Reference example 19 and the mixture was stirred at room temperature for 4 hours. Then, when a large amount of ether was added to the reaction mixture, a red-brownish oily product was precipitated. Almost all the ether layer was removed and the oily product was suspenced in chloroform. 'Che suspension was washed with a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform, The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure. White solid obtained was recrystal:Lized from n-hexane-chloroform-methanol to obtain the title compound (192 mg) .
1H-NMR (CDC13, 8): 1.7-2.1 (4H, m), 3.05 (2H, t, J=8.3Hz), 3 .53 (2H, m) , 3. 67 (2H, t, J=8 . 3FIz) , 3.75 (3H, s) , 3.89 (3H, s) , 4 .75 (1I-I, m) , 6.73 (1H, d, J=7.8Hz) , 7.16 (1H, d, J=7.8Hz), 7.47 (1H, m), 7.55 (2H, m), 8.09 (2EI, m), 8.45 (1H, s) .
Example 9 ~vnthesis Qf Na- f 1- f (2,, 4-diamin - -~t~,ridiny,l ) methyl l -, indolin-5-carbon~lLNS- (3-methoxvcarbo~lb nz 1 -ornithine methy_1 ester_ In dimethylacetamide (2.5 ml) were suspended the com-pound (192 mg) of Reference example 15 and 6-bromomethyl-2,4-diaminopteridine hydrobromide (142 mg) and the suspen-sion was stirred at 50 to 55 °C for 4 hours. After cool-ing, to the mixture was added water (5 ml) containing triethylamine (43 mg), and the mixture was stirred and then extracted with chloroform. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue obtained was applied to silica gel chromatography by using a mixed solvent of chloroform : methanol = 10 : 1 as an eluent to obtain the title compound (110 mg).
1H-NMR (CDC13, 8) : 1 .7-2.1(4H, m) , 3.06 (2H, t, J=8.3Hz) , 3.55 (4H, m), 3.78 (3H, s), 3.93 (3H, s) , 4 .52 (2H, s) , 4 .79 (1H, m) , 6.51 ( 1H, d, J=7 . 8Hz ) , 7 . 05 ( 1H, d, J=7 . 8Hz ) , 7.9-7.7 (3H, m), 8.0-8.2 (2H, m), 8.45 (1H, m), 8.77 (1H, s).

E x amp 1~.1_Q
Synthesis of N,a-_~1 _f (?, 4-diamin~fz--p~eridinyl) meth .yl l -in din ~-ca.rbonvl~~NS-isophthaloyl.-ornithing The compound (110 mg) of Example 9 was dissolved in ethanol (19 ml), and 1N-sodium hydroxide aqueous solution (0.48 ml) was further added to the solution and the mixture was stirred at 35 °C for 4.5 hours. After the mixture was stirred at 25 °C for 20 hours, water (1 ml) was added t o the reaction mixture and the reaction mixture was evaporat-ed to dryness under reduced pressure. During this proce-dure, outer temperature was so controlled not to exceeding 30 °C. A yellowish solid material was obtained and dissolved in water (5 ml), and the solution was adjusted to pH=3.7 with 1N-hydrochloric acid and allowed to stand in a refrigerator for 2 hours. Deposited precipitates were collected by filtration to obtain the title compound (78 mg ) .
1H-NMR (DMSO-d6, 8) : 1 . 5-2 . 0 (4H, m) , 3. 00 (2H, t, J=
8.8Hz), 3.32 (2H, m), 3.59 (2H, t, J=8.8Hz), 4.90 (1H, m), 9.56 (2H, s), 6.6.9 (1I-I, d, J=8.3Hz), 7.56 (1H, m), 7 . 63 (2H, m) , 8 . 0-8 . 2 (2H, m) , 8. 94 (1H, s) , 8 . 65 (1H, m) , 8.73 (1H, s) .
Reference example 16 Synthesis of Ns-(4-methoxycarbonylbenzoyl)-Na-carbo-benzoxy-ornithine methyl ester To a dichloromethane (60 ml) solution of Na-carbo-benzoxy-L-ornithine (2.0 g) was added methyl terephthalate chloride (3.0 g), and then, water (60 ml) and potassium carbonate (4.8 g) were added to the mixture and the mixture was stirred at room temperature for 15 hours. The mixture was condensed under reduced pressure to 50 ml and adjusted to pH=3 with 1N-hydrochloric acid, and deposited precipi-tates were collected by filtration and vacuum dried. White solid obtained was dissolved in a low water-content methan-ol (100 ml) and the solution was cooled to -30 °C and stir-~~~rs~
red for 10 minutes. Then, t hi.onyl chloride (3 m1) was graclually added dropwise at the same temperature. The reaction solution was slow:Ly returned to room temperature and refluxed for further 2 hours. The solvent was removed under reduced pressure and the residue obtained was subjected to silica gel chromatography by using chloroform . methanol. = 100 . 1 as an eluent to obtain the title compound (710 mg).
1H-NMR (CDC13, b): 1.6-2.1 (4H, m), 3.51 (2H, m), 3.74 (3H, s), 3.94 (3H, s), 4.43 (1H, m), 5.11 (2H, s) , 5.53 (1I-I, m) , 6. 63 (1H, bs) , 7.34 (5H, s), 7.83 (2H, d, J=8.8Hz), 8.08 (2H, d, J=8.8Hz) .
Reference example 17 synthesis of Ns. !~-methoxycarbonylbenzovl~ orn9.i<hi.n~
methyl ~yt~r, After adding a 10 b palladium-carbon (100 mg) to a methanol solution (100 ml) of the compound (710 mg) of Reference example 16, the mixture was stirred under hydro-gen atmosphere at room temperature for 20 hours. Pallad-ium-carbon was removed by filtration using Celite and the solvent was removed under reduced pressure. The residue obtained was subjected t o silica gel chromatography by using chloroform : methanol = 100 : 3 as an eluent to obtain the title compound (410 mg).
1H-NMR (CDC13, b): 1.6-2.1 (4H, m), 3.49 (3H, m), 3.73 (3H, s) , 3. 94 (3F-I, s) , 7 .20 (1H, m) , 7 .84 (2H, d, J=8.3Hz) , 8 .09 (2H, d, J=8.3Hz) .
Referens:e exampl~l~
.synthesis of Nab-carbobenzoxyindolin-5-carbonyl)-NS_, (4-methoxycarbonylbenzoyl)-ornithine methyl ester By adding thionyl chloride (2.5 ml) to 1-carbobenzoxy-indolin-5-carboxylic ac:Ld (260 mg) to prepare suspension, and a catalytic amount of di.methylformamide was further added to the suspension and the mixture was stirred at room 'temperature for 2 hours. Then, the mixture was evaporated ~~~S~.4cu~i~
to dryness under reduced pressure. The .resul.t.ing solid material was dissolved in dich:Lo.romethane (6 ml), and to the solution were added the compound (245 mg) of Reference example 17 and potassium carbonate (812 mg). Then, water (6 ml) was further added thereto, and the mixture was vigorously stirred at room temperature for 12 hours. Next, the reaction mixture was poured into water and extracted with chloroform, and the chloroform layer was washed with 1N-hydrochloric acid and dried over sodium sulfate. The solvent was removed under reduced pressure. The residue obtained was subjected t o silica gel chromatography using chloroform : methanol = 100 . 3 as an eluent to obtain the title compound (310 mg).
1H-NMR (CDC13, 8) : 1 .7-2.2 (4H, m) , 3. 16 (2I-I, t, J=8.3Hz) , 3. 61 (1I-I, m) , 3.79 (3H, s) , 3. 94 (3I-I, s) , 4 . 11 (2I-1, t, J=8 .3Hz) , 4 .83 (1H, m) , 5.29 (2H, s) , 6.89 (1H, d, J=7 . 3I-Iz) , 7. 02 (1H, m) , 7 .40 (5H, m) , 7. 66 (2I-i, d, J=7 .3Hz) , 7 . 90 (2H, d, J=8 . 8Hz) , 8.08 (2H, d, J=8.8Hz) .
Reference example 19_ Synthesis of Na-(indolin-5-carbonyl)--NS-(4-methoxy-~arbonylbenzoyl)-ornithine methyl ester To a 30 % hydrogen bromide-acetic acid (10 ml7 solu-tion of phenol (1.0 g) was added the compound (400 mg) of Reference example 18 and the mixture was stirred at room temperature for 4 hours. Then, when a large amount of ether was added to the reaction mixture, a red-brownish oily product was precipitated. Almost all the ether layer was removed and the oily product was suspended in chloroform. The suspension was washed with a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure. White solid obtained was recrystallized from n-i:~ ~3 ~ ~a ~ P~
hexane-chloro f.orm--methanol to obtain the title cornpound (145 mg) .
lI-I-NMR (CDC13, &) : 1 . 6-2. .1 (4H, m) , 3. 05 (2H, t, J=8 . 3Hz) , 3.48 (2I-I, m) , 3. 62. (2I-I, t, J=8.3Hz) , 3.76 (3H, s), 3.94 (3H, s), 4.75 (1H, m) , 6 . 57 ( 1H, d, J=7 . 8Hz ) , 7 .19 ( 1H, d, J=7 . 8flz) , 7.55 (2H, m) , 7 . 89 (2H, d, J=8.3Hz) , 8.07 (2II, d, J=8.3Hz) .
Example 11 ~mthesis of Na=L1-f(2,4-diami.no-6-~teridinyl)methyll-indolin-5-carbonyl)-NS ~4-methoxycarbonylbenzoyl)-ornithine methyl ester In dimethylacet amide (2.0 ml) were suspended the com-pound (140 mg) of Reference example 19 and 6-bromomethyl-2,4-diaminopteridine hydrobromide (105 mg) a.nd the suspen-sion was stirred at 50 to 55 °C for 4 hours. After cool-ing, to the mixture was added water (4 ml) containing triethylamine (32 mg), and the mixture was stirred and then extracted with chloroform. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography by using a mixed solvent of chloroform : methanol = 10 . 1 as an eluent to obtain the title compound (180 mg).
1H-NMR (CDC13, 8) : 1 . 6-2 .1 (4H, m) , 3. 08 (2H, t, J=8.8I-Iz) , 3.4-3.7 (4H, m), 3.79 (3H, s), 3.94 (3H, s), 4.54 (2H, s), 4.83 (1H, m), 6.52 ( 1H, d, J=8 . 3F-Iz ) , 6 . 7 9 ( 1H, d, J=6 . 8Hz ) , 7.17 (1H, m), 7.59 (2H, m), 7.92 (2H, d, J=8.3Hz), 8.09 (2H, d, J=8.3Hz), 8.81 (1H, s) .
Example 12 SSrnthesis of Na-f1-f12,4-diamino-6-pteridinyl)methyll-indolin-5-carbonyl)-Ns-teleisophthaloyl-Qrnithine The compound (150 mg) of Example 11 was dissolved in ethanol (24 m1), and 1N-sodium hydroxide aqueous solution (0.8 ml) was fu.rt~h<~r added to the solution and the rn.ixture was stirred at 35 °C fo:c 4.5 hours. After the mixture was stirred at 25 °C :~'o.r 20 hours, water (1 ml) was addecl to the reaction mixture and the reaction mixture was evaporat-ed to dryness under reduced pressure. During this proce-dure, outer 'temperature was so cone rolled not to exceeding 30 °C. Yellowish solid material obtained was dissolved in water (5 ml), and the solution was adjusted to pH=3.7 with 1N-hydrochloric acid and allowed to stand in a refrigerator for 2 hours. Deposited precipitates were collected by filtration to obtain 'the title compound (1.09 mg) .
1H-NMR (CDC13, &): 1.5-2.0 (4H, m), 3.00 (2H, t, J=8.8Hz), 3.30 (2I-I, m) , 3.58 (2H, t, J=8 . 8Hz) , 4.36 (1H, m) , 4.54 (2H, s) , 6. 68 (1H, d, J=8.8Hz), 7.62 (2H, m), 7.91 (2H, d, J=8.3Hz), 7.99 (2H, d, J=8.3Hz), 8.10 (1.H, d, J=7.8I-Iz), 8.61 (1H, m), 8.71 (1H, s) .
mp; 215-220 °C (decomposed) Reference example 20 Synthesis of ~c-benzyl N-t-butoxycarbonyl-~y-anilido-L-glutamate To a tetrahydrofuran (8 ml) solution of (x-benzyl N-t butoxycarbonyl-L-glutamate (2.2 g) and triethylamine (0.92 ml) was added a t etrahydrofuran (2 ml) solution of isobutyl chlorocarbonate (0.8 ml) at -20 °C under nitrogen atmos-phere and the mixture was stirred for 30 minutes. Then, aniline (0.5 ml) was added to the mixture, and the mixture was stirred for one hour. The mixture was returned slowly to room temperature and stirred for further 20 hours. The solvent was removed under reduced pressure and the residue obtained was dissolved in chloroform. The chloroform layer was washed with a saturated sodium hydrogen carbonate aque-ous solution, dried over sodium sulfate and then condensed under reduced pressure. The residue obtained was applied ~~.~l~u~~
to silica gel ch:romatograptry by using chloroforrn : rnet:hanol.
- 99 . 1 as an eluent to obtain the tit:l.e compound (1. 7 g) .
1H-NMR (CDCl~, &) : 1..44 (9fI, s) , 1 . 79-1 . 9'7 (1H, m) , 2.27-2.40 (2H, m), 4.32--9.44 (1H, m), 5.16 (2H, s) , 5.22-5.38 (1I-I, m) , 7 .08 (1H, t, J=7 .3Hz) , 7.26-7.33 (7I-I, m) , 7 .55 (2H, d, J=7 . 8Hz) , 8 .42 (1I-I, s) .
Reference example 21 Synthesis of a-b~nzyl ~~-anilido-L-Glutamate The compound (1.7 g) of Reference example 20 was dis-solved in trifluoroacetic acid (8 ml) under ice cooling and the mixture was stirred for 30 minutes. The solvent was removed under reduced pressure and the residue was dissolv-ed in chloroform. The chloro form layer was washed with a saturated sodium hydrogen carbonate aqueous solution and then dried over sodium sulfate. The solvent was removed under reduced pressure to obtain the title compound (1.3 g) .
1H-NMR (CDC13, b): 1.86-2.00 (1H, m), 2.17-2.38 (1H, m), 2 . 42-2 .53 (2I-I, m) , 3.53-3. 60 (1H, m) , 5.15 (2H, s), 7.07 (1H, t, J=7.3Hz), 7.29-7.35 (7H, m), 7.48 (2H, d, J=7.8Hz) , 8.27 (1H, bs) .
Reference exam~,le 22 ~ynthesi~of a-benzyl N-(1-carbobenzoxvindolin-5-carb9nyl)-~ anilido-L-glutamate Thionyl chloride (2 ml) was added to 1-carbobenzoxy-indolin-5-carboxylic acid (419 mg) and the mixture was stirred at room temperature for 2 hours. Then, the mixture was evaporated to dryness under reduced pressure. Solid product obtained was dissolved in dichloromethane (2 ml) and to the solution was added dichloromethane (2 ml) solution containing the compound (400 mg) of Reference example 21 and triethylamine (0.21 ml) under ice cooling and nitrogen atmosphere, and the mixture was stirred over-night. The reaction mixture was washed successively with - 37 _ 1N-hydroch:Lorir_ acid, a saturated sodium hydrogen carbonate aqueous solution and water, dried over sodium sulfate, and then 'the solvent was removed under :reduced pressure. The residue obtained was subjected to silica gel chromatography using, as el.uents, dichloromethane and then chloroform to obtain the title compound (317 mg).
ZH-NMR (CDC13, 8) : 2.. 10-2.20 (1H, m) , 2.29-2 .51 (3H, m) , 3.05 (2H, t, J=8.8Hz), 4.06 (2H, t, J=
8.8Hz), 4.76-4.88 (1H, m), 5.18 (2H, s), 5.28 (2H, bs), 7.05 (1H, t, J=7.3Hz), 7.18 (1.I-I, d, J=7.8I-Iz), 7.23-7.30 (2H, m) , 7 . 33 (5H, s) , 7 . 39 (5I-I, s) , 7 .43-7 . 66 (4I-I, m) , 7 .80-7 . 84 (4H, m) , 8 .57 (:LH, bs) .
Reference exampl 2 synthesis of N- (1.--carbobenzoxyin~lolin-5-carbonyl) -,~nilido-L-glutamiC acid, The compound (580 mg) of Reference example 22 was dissolved in a mixed solvent (30 ml) of chloroform methanol = 1 . 2, and 1N-sodium hydroxide aqueous solution (0.98 ml) was added thereto and the mixture was stirred at room temperature overnight. While maintaining the water bath temperature to 30 °C or lower, the solvent was removed under reduced pressure. The residue obtained was dissolved in water and after the solution was made acidic with 1N-hydrochloric acid, the mixture was extracted by using chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was removed under reduced pressure to obtain the title compound (413 mg).
zH-~IMR (CDC13, b) : 2 .17-2.29 (2H, m) , 2.52-2.56 (2H, m) , 2 .79 (2H, t, J=7. 8Hz) , 3.81 (2H, t, J=
7.8Hz), 4.51-4.61 (1H, m), 5.17 (2H, s), 6.94 (1H, t, J=7.3Hz), 7.13 (2H, t, J=7.8Hz), 7.34 (5H, s), 7.43-7.65 (5H, m) , 8. 03 (1H, bs) , 9 .02 (1H, s) .
Reference example 24 - 38 _ ~~3~5/U~ ~~J' ~~1?~~~f...~l~~Y:1 N~L1~~~~~~.l:inclol.in-~.
Vinyl)-y-ani_1_i -L~alu~ am_att:
The compound (470 mg) of Reference examp~.e 23 was dissolved in dried methanol (20 ml), and tri.methylsilyl-diazomet.hane (2 ml) was added to the solution and the mixture was stirred for 10 hours. Trimethylsilyldiazo-methane (3 ml) was further added to the mixture and the mixture was stirred for 20 hours. After removing the solvent under reduced pressure, the residue obtained was subjected to silica gel chromatography by using, as eluents, chloroform and then chloroform : methanol = 199 :
1 to obtain the title compound (252 mg).
1H-NMR (CDC13, b) : 2. 06-2.21 (1I-1, m) , 2 . 31-2 .56 (3H, m) , 3. 08 (2H, 't, J=8. 8F~Iz) , 3. 77 (3H, s) , 4 .08 (2I-I, t, J=8.8Hz) , 4.77-4 .87 (1H, m) , 5.28 (2H, s) , 7 . 07 (1H, t, J=7.3I-Iz) , 7.15 (1H, d, J=7.8Hz), 7.2_8 (2H, t, J=7.8Hz), 7.37-7.43 (5H, m), 7.58 (2H, d, J=7 . 8Hz) , 7 . 62-7 . 68 (2I~I, m) , 7 . 86 (1H, bs) , 8. 67 (1H, bs) .
Reference exam-ple 25 Synthesis of a-methyl N-(indolin-5-carbonyl)-anilido-L-Glutamate After adding 10 o palladium-carbon (50 mg) to a methanol solution (10 m1) of the compound (250 mg) of Ref-erence example 24, the mixture was stirred under a hydrogen atmosphere at room temperature for 15 hours. Palladium-carbon was removed by filtration using Celite, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography using, as eluents, chloroform : methanol = 99 : 1 and then chloroform : methanol = 19 . 1 to obtain the title compound (172 mg) .
1H-NMR (CDC13, 8) : 1 . 99-2.13 (1I-I, m) , 2.36-2.55 (3H, m) , 3.03 (2H, t, J=8 . 8Hz) , 3. 64 (2H, t, J=
8.8Hz), 3.77 (3H, s), 4.08 (1H, bs), 4.79-!1.88 (1H, m), 6.55 (1H, d, J=
8.3Hz), 6.92 (1H, d, J='7,3Hz), 7.08 (1H, t, J=7 . 3Hz ) , 7 . 30 (2i-i, t, J=7 . 8Hz ) , 7.53-7 . 65 (4H, m) , 9.01 (1i-I, bs) .
Example 1~
Synthesis of a-methyl N-f1-f(2~4-diamino-6-pteridin~
yl. ) methyl l -~indolin-5-carbonyl 1 -~~-anilido-C~-glutamate, In dimethylacetamide (5 ml) were suspended the com-pound (169 mg) of Reference example 25 and 6-bromomethyl.-2,4-diaminopteridine hydrobromide isopropanol adduct (223 mg), and the suspension was stirred at room temperature for 24 hours. The reaction mixture was applied to silica gel chromatography by using, as eluents, ethyl acetate and then chloroform : methanol = 19 . 1 to obtain the title compound (82 mg) .
1H-NMR (CDC13, 8): 2.01-2.09 (:lH, m), 2.39-2.48 (3H, m), 3. 04 (2F-1, t, J=8. 8Hz) , 3.58 (2H, t, J=
8. 8I-Iz) , 3.78 (3H, s) , 4 .53 (2H, s) , 4.79-4.89 (1H, m), 6.48 (1I-I, d, J=
7.8Hz), 6.87 (1.H, d, J=7.8Hz), 7.08 (1H, t, J=7.8Hz), 7.26-7.32 (2H, m), 7.59-7.64 (4H, m) , 8.81 (1H, s) .
Example 14 Synthesis of N-f1-f(2,4-diamino-6-pteridin~l)methvll-indolin-5-carbonyll-y-anilido-L-alutamic acid The compound (82 mg) of Example 13 was dissolved in a mixed solvent (15 ml) of ethanol . water = 2 . 1, and 1N-sodium hydroxide aqueous solution (0.15 ml) was further added to the solution and the mixture was stirred at room temperature far 60 hours. While maintaining the tempera-ture of the water bath 30 °C or lower, the solvent was removed under reduced pressure. The residue obtained was dissolved in a saturated aqueous sodium hydrogen carbonate solution and the solution caas adjusted to pH=3.7 with 1N-hydrochloric acid, Deposited orange precipitates were ~~c~~~~i.
collected by filtration to obtain the title compound (61 mg ) .
1H-NMR (DMSO-d6, b): 1.99-2.23 (2H, m), 2.42-2.51 (2H, rn), 2.98 (2H, t, J=7.8Hz), 3.59 (2H, t, J=
8.3Hz), 4.32-4.43 (1H, m), 4.56 (2H, s) , 6.70 (1H, t, J=7 .8I-Iz) , 6. 90 (2H, bs), 7.00 (1H, d, J=7.8Hz), 7.26 (2H, t, J=7.8Hz), 7.55-7.66 (4H, m), 7.72-7 . 85 (1H, rn) , 7 . 89-7. 97 (1H, m) , 8 .Z0 (1f3, d, J=7 .3Hz) , 8 .75 (1H, s) , 9. 93 (1H, s) .
Reference examgle 26 Synthesis of a-benzvl N-t-butoxyc~rbon_xl-.~-(2-me~h-Qxycar~o~lanilido)-L-Glutamate N,N'-carbonyldiimidazole (2f2 mg) was added to a tetrahydrofuran solution (5 ml) of a-benzyl N-t-butoxy-carbonyl-L-glutamate under ice cooling and nitrogen atmos-phere, and the mixture was stirred for one hour. Then, a .
tetrahydrofuran solution (2 ml) of methyl o-aminobenzoate (0.19 ml) was added thereto, and the mixture was stirred for 5 hours. After gradually returning the temperature~to room temperature, the mixture was stirred for a further 48 hours. The solvent was removed under reduced pressure, and the residue obtained was applied to silica gel chromatogra-phy using chloroform as an eluent to obtain the title compound (273 mg) .
1H-NMR (CDC13, 8): 1.41 (9H, s), 2.04-2.18 (1H, m), 2.20-2.32 (1H, m), 2.48-2.57 (2H, m), 3.92 (3H, s) , 4.34-4.47 , (1H, m) , 5.18 (2H, s), 5.19-5.27 (1H, m), 7.08 (1H, t, J=
7. 8Hz) , 7.34 (5H, s) , 7.53 (1H, t, J=
7.3Hz), 8.02 (1H, d, J=7.8Hz), 8.68 (1H, d, J=7.8Hz), 11.06 (1H, s) .
Reference example 27 Synth~~is of a-benzyl y-(2-methoxycarbonylanilido)-L-g_lu~ama~g The compound (632 rng) of Reference example 26 was dis-solved in tri:Eluoroacetic acid (9.5 m:L) under ice coolinrs and the mixture was stirred for 90 minutes. The solvent was removed under reduced pressure, and the residue was dissolved in chloroform. The chloroform layer was washed with a saturated aqueous sodium hydrogen carbonate solution and dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography by using, as eluents, chloro-form and then chloroform : methanol = 97 . 3 to obtain the title compound (913 mg) .
1H-NMR (CDC13, 8) : 1 . 91-2 . 02 (1I-1, rn) , 2 .20-2. 30 (1.H, m) , 2 .55-2 . 69 (2H, m) , 3. 56-3 . 62 (1H, rn) , 3. 92 (3H, s) , 5.17 (2H, s) , 7.07 (1H, t, J=8.3~Iz) , 7 .35 (5H, s) , 7 .53 (1H, t, J=
8.8Hz), 8.02 (1H, d, J=8.3Hz), 8.69 (1H, d, J=8.8Hz), 11.08 (1H, bs).
Reference example synthesis of 1-f(2~4-diamino-6-pteridinyl)methylL
indolin-5-carboxylic aci~1 In dimetylacetamide (2 ml) were suspended 1-carbo-benzoxyindolin-5-carboxylic acid (87 mg) and 6-bromomethyl-2,4-diaminopteridine hydrobromide 1/2 isopropanol adduct (136 mg), and the suspension was stirred at 55 °C for 9 hours. To the reaction mixture was added water (20 ml) and the mixture was allowed to stand in a refrigerator over-night. Precipitated solid was collected by filtration, dissolved in a small amount of 1N-sodium hydroxide aqueous solution and the solution was adjusted to pi-1=6.5 with 1N-hydrochloric acid. Deposited brownish precipitates were collected by filtration to obtain the title compound (56 mg ) .
1H-NMR (DMSO-d6, b) : 3.01 (2H, t, J=8 .8Hz) , 3. 64 (1H, t, J=
8 . 8Hz) , 9 . 60 (2H, s) , 6. 68 (1H, d, J=
8.3Hz), 7.16 (2H, bs), 7.58 (1H, s), t. t' )l~ L5 t7 L~. i3 ;:~
7. 65 (1i-I, d, J=8. 3I-Iz) , 8.09 (1H, bs) , 8 .25 (:LI-l, bs) , 8.77 (lFi, s) .
Example 15 ~~.~~~ -~--~r ~H.~1-1 N- r 1- r (1.~4~~~~.~c~~i.. 1 '~~ ~.
Y~J~.I.P.~h.Y.~..L-~ ndo in__~~carbQi~yl ~ -v- (2-methoxy~I~nYJ=
,~ni .i"~lo) --L-ahutamate To a dimethylformamide suspension (5 ml) of the com-pound (134 mg) of Reference Example 28 and 1-hydroxybenzo-triazole (108 mg) was added a dimethylformamide solution (1.5 ml) of dicyclohexylcarbodiimide (123 mg) under ice cooling and nitrogen atmosphere, and the mixture was stir-red for 30 minutes. Then, a dimethylformamide solution (1.5 ml) of the compound of Reference example 27 was added to the mixture, the mixture was gradually returned to room temperature and stirred for 24 hours. The reaction mixture was subjected to silica gel chromatography by using, as eluents, ethyl. acetate and then chloroform : met hanol = 19 . 1 to obtain the title compound (29 mg).
~H-NMR (CDC13, 8): 2.32-2.46 (2H, m), 2.96-2.70 (2H, m), 2.96 (2H, t, J=8.8Hz), 3.49 (2H, t, J=
8.3Hz), 3.89 (3H, s), 4.46 (2H, s), 4.83-4.90 (1H, m), 5.20 (2H, s), 6.40 (1F3, d, J=8. 3Hz) , 7.05 (1H, t, J=8.3Hz) , 7 .14 (1H, d, J=7 . 8Hz) , 7 .34 (5H, s) , 7.41 - 7.59 (3H,m), 7.96 (1H, d, J=8.3Hz), 8.64 (1H, d, J=8.3Hz), 8.77 (lI-I, s) Example 16 ~vnthesis of N-r1-r(2,9-diamino-6-pteridin~l)methyll in lin- -r~rbonyll -Y- t2-methox~carbon,ylanilido) -L-c~lutamic acid The compound (28 mg) of Example 15 was suspended in methanol (2.5 ml), and 1N-sodium hydroxide aqueous solution (0.41 ml) was added to the suspension and the mixture was stirred at 10 °C for 5 hours. While maintaining the tem-perature of water bath to 30 °C or lower, the solvent was r 1~~.~~~:)i~iDJ
removed under reduced pressure. The residue obtained was dissolved in a saturated aqueous sodium hydrogen carbonate solution and the solution was adjusted to p.H=3.7 with 1N-hydrochloric acid. Deposited orange precipitates were collected by filtration to obtain the title compound (21 mg ) .
xH-NMR (DMSO-d6, 8) : 1 . 95-2 .25 (2H, m) , 2 . 39-2 .58 (2H, m) , 2 . 98 (2I3, t, J==8 . 3Hz) , 3 .59 (2H, t, J=
8 . 3Hz) , 9 . 38-4 .50 (1H, m) , 4 .57 (2I-I, s) , 6. 69 (1I1, d, J=8.3Hz) , 7.12 (1H, t, J=7.8I-Iz) , 7.24-7 .35 (2H, rn) , 7 .51-7. 64 (3H, m) , 7 . 96 (1H, d, J= 7. 8I-Iz) , 8.04-8.39 (4H, m) , 8.47 (1.I-I, d, J=
7 . 8I-Iz) , 8. 78 (1H, s) , 11 .25 (2I-1, s) .
Reference example 29 Synthesi~of a-methyl 'y-k~enzyl N-t-butoxycarbon5rl-h-alutamate Tn a dimethylformamide (75 ml) solution of 'y-benzyl N-t-butoxycarbonyl-L-glutamate was suspended sodium hydrogen carbonate (2.5 g), and then a dimethylformamide (75 ml) solution of methyl iodide (10.52 g) was added to the suspension and the mixture was stirred at room temperature for 24 hours. After the reaction mixture was condensed under reduced pressure, water (70 ml) was added to the residue and the mixture was extracted with ethyl acetate n-hexane = 1 . 1 solution. The organic layer was washed with water and dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography by using ethyl acetate : n-hexane = 1 . 2 as an eluent to obtain the title compound (5.2 g).
1.H-NMR (CDC13, b) : 1 .43 (9H, s) , 1 . 9?.-2. Ol (1H, m) , 2 . 11-2 . 37 ( 1H, m) , 2 . 42-2 .51 (2H, m) , 3.73 (3H, s), 4.23-4.40 (1H, m), 5.12 (2H, s) , 7 . 35 (5H, s) .
Reference example 30 ~i~~tS~~~r s~Y~l.the~a-S Q ~~~ .C~~~.rlyi_...~1 ~r>~..k~~~2~.Y~~.liatamaLa After adding 10 0 ,palladium-carbon (1.1 g) to a methanol solution (30 ml) of the compound (5.2 g) of Refer-ence example 29, the rni.xture was stirred under hydrogen atmosphere at room temperature for i5 hours. Palladium-carbon was removed by filtration using Celite and the solvent was removed under reduced pressure. The residue obtained was dissolved in a saturated aqueous sodium hydrogen carbonate solution and the solution was washed with chloroform. After separating the aqueous layer, the aqueous layer was adjusted to pH=4 with 5 % citric arid and then extracted with chloroform. The chloroform layer was dried over sodium sulfate and the solvent was removed under reduced pressure to obtain the title compound (3.9 g).
1H-NMR (CDC13, S) : 1.44 (9H, s), 1.89-2.04 (1I-I, m), 2.09-2.27 (1H, m), 3.75 (3H, s), 4.34 (1H, m) , 5. 17-5.21 (1II, m) , 9.38 (1H, bs) .
Reference example 31 synthesis of ~x-methyl N-t-butox~carbon~l-v- l3-ethoxy-carbonylanilido)-L-glutamatg To a dichloromethane suspension (10 ml) of the com-pound (1.6 g) of Reference example 30 and 1-hydroxybenzo-triazole (0.8 g) was added a dichloromethane solution (5 ml) of dicyclohexylcarbodiimide (1.5 g) under ice cooling and nitrogen atmosphere, and the mixture was stirred for 30 minutes. Then, ethyl m-aminobenzoate (1.5 g) was added thereto, the mixture was gradually returned to room tem-perature and stirred for 20 hours. The solvent was removed under reduced pressure and t o the residue obtained was added ethyl acetate, and white insolubles were filtered off. The filtrate was condensed under reduced pressure and the residue obtained was subjected to silica gel chromato-graphy by using, as eluents, chloroform and then chloroform . methanol = 99 : 1 to obtain the title compound (2.5 g).
1H-NMR (CDC13, 8) : 1 .38 (3H, t, J=7.lHz) , 1 .47 (9H, S) , 1.82-2.04 (1H, m), 2.20-2.39 (1H, m), ~~S~i~J~~
2 .45-2 .51 (2I-I, rn) , 3.74 (3H, s) , 4 . 32-4 . 43 (1H, m) , 4 .37 (2Ii, q, J=7 . 1Hz) , 5.37 (1.H, bd, J=7.3Hz) , 7.40 (1H, t, J=7 . 8EIz) , 7.78 (1H, d, J=7 . 8Hz) , 7 . 96 (1H, d, J=9.3H:z) , 8.13 (1H, s) , 8.83 (1H, bs) .
Reference example 32 Synthesis ~~~-me~hvl ~y-y3-ethoxycarbon~y~nilido)-L-alut amat e.
The compound (2.5 g) of Reference example 31 was dis-solved in trifluoroacetic acid (15 ml) under ice cooling and the solution was stirred for one hour. The solvent was removed under reduced pressure and the residue was dissolv-ed in chloroform. The chloroform layer was washed with a saturated aqueous soda.um hydrogen carbonate solution and dried over sodium sulf ate, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromat agraphy by using chloroform: methanol - 19 . 1 as an eluent to obtain 'the 'title compound (1.6 g).
1H-NMR (CDC13, 8) : 1 .39 (3H, t, J=7.lHz) , 1 .74 (2H, s) , 1.82-1.99 (1H, m), 2.15-2.32 (1H, m), 2.52-2 . 62 (2H, m) , 3.54-3. 60 (1H, m) , 3.74 (3H, s), 4.37 (2H, q, J=7.lHz), 7.39 (1H, t, J=7.8Hz), 7.77 (1H, d, J=7.3Hz), 7.94 (1H, d, J=7.8Hz), 7.99 (1H, s) , 8. 63 (1H, bs) .
Reference example 33 Synthesis of a-methyl. N-(1-carbobenzox,yindolin5-5-carbonyl) -~ (3-ethoXVCarbon~lanilido) -L-gl~amate Thionyl chloride (4 ml) was added to 1-carbobenzoxy-indolin-5-carboxylic acid (612 mg), and the mixture was stirred at room temperature for 2 hours. Then, the mixture was evaporated to dryness under reduced pressure. Solid material obtained was dissolved in dichloromethane (4 ml) and to the solution was added a dichloromethane solution (4 ml) containing the compound (529 mg) of Reference example ~~~S~p~)J
32 and trietYiylamine (0,:36 ml) under. ice cooling and vitro--gen atmosphere, and the rn:ixtu.re was stirred overnight. The reaction mixture was successively washed with 2Di-hydro-chloric acid, a saturated aqueous sodium hydrogen carbonate solution and water, and dried over sodium sulfate, and then the solvent was removed under. reduced pressure. The resi-due obtained was applied to silica gel chromatography by using, as eluents, chloroform and 'then chloroform methanol = 99 : 1 to obtain the title compound (870 mg).
1H-NMR (CDC13, b) : 1 . 37 (3H, t, J=7 ,1Hz) , 2 . 08-2 .22 (1H, m) , 2, 36-2.57 (3II, m) , 3.06 (2H, t, J=
8. 8Hz) , 3.77 (3I-I, s) , 4 . U7 (2H, t, J=
8 . 8Hz) , 4 .30-4 . 40 (2I3, q, J=7. 1Hz) , 4.78-4.87 (1H, m), 5.28 (2H. bs), 7.13 ( 1I3, d, J=7 . 8I3z ) , 7 , 30-7 . 4 3 ( 6H, m) , 7.60-7.67 (2H, m), 7.74 (1H, d, J=
7.8I-Iz) , 7.89 (1H, d, J=7. 8Hz) , 8.12. (1H, bs) , 8. 96 (1H, bs) .
Reference example 34 Synthesis of a-methyl N-(indQlin-5-carbonyl)-~-(3-~h,Qx ar onylanilido)-L-glutamate After adding 10 % palladium-carbon r0.17 g) to a methanol solution (11 ml) of the compound (892 mg) of Ref-erence example 33, the mixture was stirred under hydrogen atmosphere at room temperature for 15 hours. Palladium-carbon was removed by filtration using Celite, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica.gel chromatography using, as eluents, chloroform and then chloroform : methanol = 99 . 1 to obtain the title compound (448 mg).
1H-NMR (CDC13, 8) : 1.36 (31-I, t, J=7.lHz) , 2.05-2..25 (1H, m), 2.25-2.48 (1H, m), 2.,48-2.59 (2H, m), 2.93 (2H, t, J= 8.8Hz), 3.57 (2H, t, J=8.8Hz), 3.71 (3H, s), 4.18 (1H, bs), 4.34 (2H, t, q, J=7.lHz), 4.73-4.84 (1H, m) , 6.46 (1H, d, J=8.8Hz) , 7.16 (1H, d, _ 47 _ J== 7 . 8Hz ) , 7 . 32 (1.I-3, t, J--7 . 8Hz) , '7 . 5U-.
7 .53 (2I-I, m) , '7 .'73 (1H, d, J=7 . SHz) , 7 . 87 (1H, d, J=7 .3f-Iz) , 8 .19 (1H, s) , 9.39 (1II, bs) .
Ex~mn~.e 17 synthesis of ~-methyl N-f1-f~2,4 ~iamino-6-pteridin-yl ) methyl l -indolin-5-Garbon~l l=~ 13~~hoxycarbonylanil ido) -L-glutamate To dimethylacetamide (5 ml) were suspended the com-pound (448 mg) of Reference Example 34 and 6-bromo-2,4-diaminopteridine hydrobromide isopropanol adduct (587 mg) and the suspension was stirred at room temperature for 36 hours. The reaction mixture was applied to silica gel.
chromatography by using, as eluents, ethyl acetate and then chloroform : methanol = 9 : 1 to obtain the title compound (653 mg) .
1H-NMR (DMSO-d6, b): 1.34 (3H, t, J=6.8Hz), 1.96-2.32 (2H, m), 2.32-2.59 (2H, m), 3.00 (2H, t, J=7.3Hz), 3.56-3.66 (5H, s), 4.32 (2H, q, J=6.5Hz), 4.40-9.56 (3H, m), 6.66-6.'71 (3H, bs) , 7 .39 (1H, t, J=7.8Hz) , 7.63-7.67 (3H, m), 7.84 (1H, d, J=
7.3Hz) , 8.22 (1H, s) , 8. 32 (1H, d, J=7.3Hz), 8.73 (1H, s), 10.11 (1H, s).
Examplee 18_ ~~mthesis of N-fl-f(2,4- iamino-6-pteridinyl~ methvll-indolin-5-carbonyll-~-r (3-r~arboxyanilido)-L-glutamic acid The compound (303 mg) of Example 17 was dissolved in methanol (10 ml), and 1N-sodium hydroxide aqueous solution (1.06 ml) was added to the solution and the mixture was stirred at room temperature for 12 hours. Water (1 ml) was added and then the mixture was further stirred for 2 hours.
While maintaining the temperature of water bath to 30 °C or lower, the solvent was removed under reduced pressure. The residue obtained was dissolved in a saturated aqueous sodium hydrogen carbonate solution and the solution was ~lf~tS~fi~i adjusted to pH=3.7 with 1N-hydrochloric acid. Deposited orange precipitates were collected by filtration to obtain the title compound (157 mg).
1H-NMR (DMSO-d6, b) : 1 . 9 7-2 .25 (2H, m) , 2 . 42-2 .5$ (2H, m) , 2. 99 (2H, t, J=8 . 8Hz) , 3 .59 (2H, t, J=
8.8Hz), 4.33-4.45 (1H, m), 4.59 (2H, s), 6.71 (1H, d, J=8.3Hz), 6.93 (2H, bs), 7.39 (1H, t, J=7.8Hz), 7.57-7.82 (5H, m), 7.88-8.04 (1H, m), 8.06-8.12 (1H, m), 8.15-8.26 (2H, m), 8.75 (1H, s) , 10. 12 (1H, s) .
Reference example 35 synthesis of a-benzyl N-carbobenzoxy-N' N' -dirnethylh-glutamiD_ate_ Ethyl chlorocarbonate (0.14 ml) was added to a tetra-hydrofuran solution (2 ml) containing a-benzyl N-carbobenz-oxy-L-glutamat a (508 mg) and triethylamine (0.19 ml) at -20 °C under nitrogen atmosphere, and tY~e mixture was stirred for 30 minutes. Then, a d.ichloromethane (3 ml) solution of dimethylamine hydrochloride (112 mg) and triethylamine (0.19 ml) was added to the mixture and the mixture was stirred for one hour. The mixture was gradually returned to room temperature,. and the residue obtained was dissolved in chloroform. The chloroform layer was washed successively with a saturated aqueous sodium hydrogen carbonate solution and 1N-hydrochloric acid, and dried over sodium sulfate, and then the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography by using chloroform : methanol =
49 : 1 as an eluent to obtain the title compound (453 mg).
~H-NMR (CDC13, 8): 1.97-2.37 (4H, m), 2.86 (3H, s), 2.90 (3H, s) , 4 .39-4 .41 (1H, m) , 5. 10 (2H, s), 5.17 (2H, s), 5.86-5.89 (1H, m), 7.34 (10H, s) .
Reference example 36 ~~a~la~~'~
n h~si~. ~.~ N.~~.'~~~~?~~~N' ~mF ;hyl.-T~-,g=lit a m i n ~, To a methanol solution (5 ml) of the compound (950 mg) of Reference example 35 was added 1D1-sodium hydroxide aque ous solution and the mixture was stirred at room tempera ture overnight. While maintaining the water bath tempera-ture to 30 °C or lower, the solvent was removed under reduced pressure. The residue obtained was dissolved in water and after the solution was made acidic with 1N-hydro-chloric acid, the mixture was extra<aed by using chloro-form. After the chloroform layer was dried over. sodiurn sulfate, the solvent was removed under reduced pressure to obtain the title compound (391 mg).
1H-NMR (CDC13, 8) : 1.. 93-2. 06 (1E1, m) , 2 .21-2. 33 (1I-I, m) , 2. 92-2 .56 (1t1, m) , 2.76-2. 89 (1f-I, m) , 2.98 (3H, s), 3.00 (3H, s), 9.19-9.27 (1I-i, m) , 5.09 (2H, s) , 6.09-6.06 (1H, m) , 7 .37 (lOH, s) .
Reference ~examplc~~ 37 ~Ynthesis of a-methyl N-carbobenzoxy-N',N'-c~im~h_SrlL-glutaminatP
In a dried methanol (9 ml) was dissolved the compound (3530 mg) of Reference example 36 anti trimethylsilyldiazo-methane (5 ml) was added to the solution and the mixture was stirred for 2 hours. The solvent was removed under reduced pressure to obtain the title compound (338 mg).
1H-NMR (CDClg, b) : 1 . 96-2.43 (4H, m) , 2. 92 (3H, s) , 2 . 95 (3H, s) , 3.79 (3H, s) , 9 .28-4.90 (1F-I, m), 5.10 (2H, s), 5.80 (1H, m), 7.35 (5H, s) .
Reference example 38 ,synthesis of a-methyl N',,N'-dimethyl-L-glutaminate After adding a 10 o palladium-carbon (70 mg) to a methanol solution (10 ml) of the compound (580 mg) of Ref-erence example 37, the mixture was stirred under hydrogen atmosphere at room temperature for 15 hours. Palladium-carbon was .removed by f:i.ltration using Celite and the solvent was removed under. reduced pressure. The residue obtained was subjected to silica gel chromatography by using chloroform : methanol = 19 : 1 as an eauent to obtain the title compound (160 mg).
1H-NMR (CDC13, 8): 1.74-1.92 (1H, m), 2.06-2.23 (1H, m), 2.47 (2H, t, J='7.3.I3z) , 2. 95 (3I-I, s) , 3. 02 (3H, s) , 3.49-3.56 (1II, rn) , 3.73 (3H, s) .
Referer~~ example 39 °wn~the~s of a-m,Qthvl N-(1-car.-bobenzox5rindcz7-ink carbonyl ) -N' , N' -dimethyl-:C.-al.utaminate Thionyl chloride (2 ml) was added t o 1-carbobenzoxy-indolin--5-carboxylic acid (2.95 mg) and the mixture was stirred at room temperature for 2 hours. Then, the mixture was evaporated to dryness under reduced pressure. Sol:i.d material obtained was dissolved in di.chlo.romethane (2 ml) and to the solution was added a dichloromethane solution (2.5 ml) containing the compound (160 mg) of Reference example 38 and triethylamine (0.18 ml) under ice cooling and nitrogen atmosphere, and the mixture was stirred over-night. The reaction mixture was successively washed with 1N-hydrochloric acid, a saturated aqueous sodium hydrogen carbonate solution and water, and dried over sodium sulfate and then the solvent was removed under reduced pressure.
The residue obtained was applied to silica gel chromato-graphy using, as eluents, chloroform and then chloroform methanol = 99 . 1 to obtain 'the title compound (359 mg).
1H-NMR (CDC13, &) : 2 .22-2 . 32 (2H, m) , 2 . 43-2 . 55 (2H, m) , 2.94 (3H, s), 2.98 (3H, s), 3.15 (2H, t, J=8.8Hz), 3.76 (3H, s), 4.10 (2H, t, J=8.8Hz), 4.58-4.68 (1H, m), 5.28 (2H, s), 7.36-7.41 (6H, m), 7.'71 (1H, s), 7.90 (1H, d, J=5.9Hz) .
Reference example 40 ~I~~~~S~(~
;~ynth~s_i~Q f (~~;. ~.L N- ~~n~iQ.l_i_r~y-carts nil L=_N!, I~I' -dimeth~l-L--=_cllu amin ~~
After adding a 1.0 ~ pa:Lladium-carbon (70 mg) to a methanol solution (5 ml) of the compound (359 mg) of Ref-erence example 39, the mixture was stirred under hydrogen atmosphere at room temperature for 15 hours. Palladium-carbon was removed by filtration using Celit-a and the solvent was removed under reduced pressure. The residue obtained was subjected t o silica gel chromatography by using chloroform : methanol = 99 : 1 as an eluent to obtain the title compound (131 mg).
1H-NMR (CDC13, b) : 2 .19-2 . 30 (2I-I, m) , 2 .94-2 .53 (2H, m) , 2 . 93 (3I-I, s) , 2 . 9 7 (3H, s) , 3. 04 (2f-I, t, J=8.3Hz) , 3. 62 (2H, t, ~!=8 .3Hz) , 3.75 (3H, s) , 4. 14 (1I3, bs) , 4. 60-4. 70 (1F-I, m) , 6.56 (1I-I, d, J= 7.8Hz) , 7.51-7. 62 (3I-I, m) .
Example 19 ~yni~hesis of a-methyl N-f1-f(2,4-diamino-6-p~eridin-yl)methyll-indolin-5-ca.rbonyll-N',N'-dimethyl-L~lutaminate In dimetylacetamide (4 ml) were suspended the compound (131 mg) of Reference example 40 and 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (233 mg), and the suspension was stirred at room temperature for 24 hours. The reaction mixture was applied to silica gel chromatography by using, as eluents, ethyl acetate and then chloroform : methanol = 9 : 1 to obtain the title compound ( 95 mg) .
1H-NMR (CDC13, 8) : 1 . 98-2 . 17 (2H, m) , 2 . 42 (2H, t, J=
6. 8Hz) , 2 . 84 (3H, s) , 2 . 94 (3I-I, s) , 3. 00 (2H, t, J=8 . 3I-Iz) , 3.59 (2H, t, J=8.3Hz) , 3.64 (3H, s), 4.30-4.42 (1H, m), 4.55 (2H, s), 6.68 (1H, d, J=7.3Hz), 7.60-7.64 (2H, m), 8.34 (1H, d, J=7.3Hz), 8.71 (1H, s) .
Example 20 ~~~~~~i~o Synth~~si~ _of N~f 1.~ f (2 ~ 4-diamino-6=pteri~inyl ) m~~)zYl1 -indolin-5-carbonyl L-~' ,, N' -dime~h_y~l.-L~-alutamine The compound (70 mg) of Example 19 was dissolved in methanol (5 ml) and to the solution was added 1N-sodiurn hydroxide aqueous solution (0.15 ml), and the mixture was stirred at room 'temperature for 12 hours. Water (2 ml) was added thereto and the mixture was further stirred for 5 hours. While maintaining the water bath temperature to 30 °C or lower, the solvent was removed under reduced pres-sure. The residue obtained was dissolved in a saturated aqueous sodium hydrogen carbonate solution and the solution was adjusted to pH=3.7 with 1N-hydrochloric acid. Deposit-ed orange precipi'ta'tes were collected by filtration to obtain the title compound (16 mg).
1H-NMR (DMSO-d6, 8) : 1 . 96--2 .08 (2H, m) , 2 .41 (2H, t, J---7 .lHz) , 2 . 82 (3H, s) , 2. 92 (3H, s) , 3. 01 (2H, t, J=7.8Hz) , 3. 62 (2H, t, J=7.8Hz) , 4 .27-4 .37 (1H, m) , 4 . 62 (2H, s) , 6.72 (1H, d, J=8.8Hz), 7.61-7.64 (2H, m), 8.24 (1H, d, J=6.8Hz), 8.87 (1H, s), 12.46 (1H, bs).
Reference example 41 Synthesis of f1-f(2,4-diamino-6-~teridinyl meth~ll 1,2,3,4-tetrahydro-6-quinolinecarboxylic acid In dimethylacet amide (3 ml) were suspended 6-bromo-methyl-2,4-diaminopteridine hydrobromide~isopropanol adduct (178 mg) and 1,2,3,4-tetrahydro-6-quinoli.necarboxylic acid (55 mg), and the suspension was stirred at 60 to 65 °C
overnight. After cooling, water (10 ml) was added to the reaction mixture, arid the mixture was adjusted to pH=3.5 with 1N-hydrochloric acid under ice-water cooling and allowed t o stand at cool place overnight. Deposited precipitates were collected by filtration to obtain the title compound (70 mg).
1H-NMR (DMSO-dg, b) : 2.08 (2H, t, J=8Hz) , 2.88 (2H, t, J=
8Hz), 3.68 (2H, t, J=8Hz), 4.72 (2H, s) , 6. 62 (lf-I, d, ~f=9I-Iz) , 7.48 (2HI, rn) , 8.51 (1H, s) .
Example 21_ synthesis of diethyl N-,j1-f(2"4-diamino-6-oteridinyl)-methyl l 1, 2 F 3 , 4-t ~~rahydro-., -aui_nol:W ecarbonyl 1 -L~qlutamate In anhydrous dimethylformamide (6 ml) were suspended triethylamine (60 mg) and diethylphosphorcyanidate (98 mg), and the compound (60 mg) of Reference example 41 was further added thereto and the mixture was stirred. After dissolving, the mixture was stirred at 80 °C for 3 minutes, and returned to room temperature, followed by stirring for 10 minutes. Then, triethylamine (20 mg) and diethyl glutamate hydrochloride (40 mg) were added t o the mixture and the mixture was stirred at 80 °C ;Por 2 hours. After the reaction, the so).vent was removed under reduced pres-sure, to the residue was added chloroform and the chloro-form layer was washed with a saturated aqueous sodium bicarbonate solution. After drying the chloroform layer over magnesium sulfate, the solvent was removed under reduced pressure and the residue obtained was applied to silica gel chromatography by using a mixed solvent of chloroform : methanol = 10 . 1 as an eluent to obtain the title compound (50 mg) .
1H-NMR (DMSO-dg, 8): l.l-1.4 (6H, m), 1.8-2.4 (4H, m), 2.40 (2H, t, J=8Hz), 2.82 (2H, t, J= 8Hz), 3.51 (2H, t, J=SHz) , 4 . 0-4 .3 (4H, m) , 4 . 64 (1H, m) , 4 .75 (2H, s) , 6 . 65 (1H, d, J=9Hz) , 7.47 (2H, m) , 8. 6 5 (1H, s) .
Example 22 Synthesis of N-f1-f(2,4-diamino-6-pteridin~l)methyll 1 ~2, 3, 4-tetrahydro-6 guinolinecarbonyl l -L-alut:amic acid In ethanol (10 ml) was dissolved the compound (50 mg) of Example 21, and to the solution was added 1N-sodium hydroxide aqueous solution (0.24 ml) at 35 °C, and the mixture was stirred at the same temperature for 4.5 hours.
Stirring was further continued at 25 °C for 20 hours, and water (1 ml) was added to the reaction mixture. Under ice-water cooling, the reaction mixture was adjusted to pH---3.7 with 1N-hydrochloric acid and allowed to stand at cool place overnight. Deposited precipitates were collected by filtration to obtain the title compound (27 mg).
~H-NMR (DMSO-ds, $) : 1 . 8-2.2 (4H, m) , 2 .31 (2H, t, J=8f-Iz) , 2 .75 (2H, t, J==8Hz) , 3. 60 (2H, t, J=
8Hz), 4.36 (1H, m), 4.70 (2H, s), 6.62 ( 1FI, d, J=9Hz ) , 7 . 51 ( 2I-I, m) , 8 . 62 (1H, s) .
mp; 204-208 °C (decomposed) Reference example 42 Svnt_hesis of methyl 4-~~2, 4-diamino-6-pteri.di~lL, methvll-3,4-dihydro-2H-1,4-benzoxazin-7_carboxylat~
In dimethylacetamide (l0 ml) were suspended 6-bromo-methyl-2,4-diaminopteridine hydrobromide°isopropanal adduct (410 rng) a.nd methyl 3,4-dihydro-2H-1,4-benzoxazin-7-carbo-xylate (200 mg), and 'the suspension was stirred at a bath temperature of 65 °C for 4 hours and at 90 °C for 19 hours.
After cooling, dimethylacetamide was condensed under reduc-ed pressure, and chloroform and an aqueous sodium hydrogen carbonate solution were added to the condensate. After precipitates were fi~.tered off, the organic layer was washed with water and dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography and eluted by using a mixed solvent of chloroform : methanol = 97 . 3 to obtain the title compound (100 mg).
1H-NMR (CDClg : CDgOD = 9: 1, $): 3.63 (2H, brt), 3.84 (3H, s), 4.32 (2H, brt), 4.72 (2H, s), 6.68 (1H, d, J=9.OHz), 7.47 (1H, s), 7.50 (1H, d, J=9.OHz), 8.71 (1H, s).
Reference example 93 ~5mthesis of 4-f (2,4-di.amino-6-pteridinyl)methyll-3,4-dihydro-2H-1,4-benzoxazin-7-carboxylic acid ~~~i~~1~3~
The compound (60 rng) of Reference example 42 was sus-pended in a mixed solvent of 1N-sodium hydroxide aqueous solution (20 ml) and methanol (20 ml) and the suspension was refluxed for 2.5 hours under heating. After cooling, the solvent was removed and water was added and the mixture was adjusted 'to pH=5 (suspended) with 1N-hydrochloric acid.
It was allowed t o stand at cool place, deposited precipitates were collected by filtration and dried 'to obtain the title compound (60.8 rng) .
1H-NMR (DMSO-d6, 8) : 3 . 63 (2I-I, rn) , A . 22 (2H, m) , 4 . 71 (2H, s), 7.21 (1H, s), 8.29 (1H, s).
Example 23 Syn~hesi,_s of dieth~r,l N- f 4- f (2, 4-diamino-6-p,~.eri. in~l~
meth~l.1 -3, 4-dihvdr_o-2H-7!. , 4-benzoxazin- 7-carbonyl l -L-alu~:amate To a solution of diethylphosphorcyani.date (64 ~.l), t riethylamine (60 ~.l) and anhydrous dimethylformamide (5 ml) was added the compound (50 mg) of Reference example 43 and the mixture was stirred under nitrogen atmosphere at 80 °C for 5 minutes. After cooling to the room temperature, triethylamine (20 ~1) and diethyl glutamate (34 mg) were added to the mixture and the mixture was again heated to 80 °C and stirred for 2.5 hours. After cooling, the reaction mixture was extracted with chloroform, and the chloroform layer was washed successively with a saturated aqueous sodium hydrogen carbonate solution and a saline solution.
After drying the chloroform layer over magnesium sulfate, the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel chromatography by using a mixed solvent of chloroform : methanol = 19 . 1 as an eluent to obtain the title compound (10 mg).
1H-NMR (CDC13:CD30D = 9:1, b): 1.23 (3H, t, J=6.8Hz), 1.29 (3H, t, J=6.8Hz), 3.60 (2H, m), 4.11 (2H, q, J=6.8Hz) , 9.22 (2H, q, J=
6.8Hz), 4.32 (2H, m), 4.71 (2H, s), 6.69 (1H, d, J=10.OFIz) , 7.29 (1.H, d, J=lO.OHz), 7.36 (1H, s), 8.70 (1H, s) .
Example 24 ~S~nthesis of N- f 4- f t2,. 4 diami_no-6-pteridinxl ) methyl L
~,4-dihydr -~?H~1,,4-benzoxazin-7-carbon~ll-L-qlutamic acid In ethanol (3 ml) was dissolvE:d the compound (9 mg) of Example 23, and t o the solution wa~~ added 1N-sodium hydr-oxide aqueous solution, and the mixture was stirred at room temperature for 4 days. The solvent was removed and the residue was subjected to silica gel column chromatography by using a mixed solvent of chloroform : methanol . aqueous ammonia = 5: 4 . 1. Fractions containing the title com-pound were collected and the solvent was removed therefrom.
The reside was dissolved by adding a saturated aqueous sodium hydrogen carbonate solution (200 ~l) and the solu-tion was adjusted to pH about 4 (suspended) by dropping 1N-hydrochloric acid and allowed to stand at cool place over-night. Deposited precipitates were collected by filtration to obtain the title compound (2.8 mg).
1H-NMR (DMSO-ds, 8): 1.86-1.95 (1H, m), 1.95-2.03 (1H, m), 2.24-2.38 (2H, m) , 3. 67 (2H, t, J=
3.8Hz), 4.25 (2H, t, J=3.8Hz), 4.30 (1H, m), 4.70 (2H, s), 6.83 (1H, d, J=8.6Hz), 7.26 (1H, s), 7.30 (1H, d, J=8.6Hz), 8.09 (1H, m), 8.70 (7.H, s).
IR (KBr) v max 3464, 1642 and 1512 cm"~.
Example 25 Synthesis of methyl N-f1-f(2,4-diamino-6=pteri in 1 -methyllindolin-5-carbonyll-DL-2-amino-4-~hosphonobutyrate In anhydrous dimethylformamide (30 ml) were suspended triethylamine (272 mg) and diethyl cyanophosphonic acid (440 mg). Then, to the suspension was added 1-[(2,4-diamino-6-pteridinyl)methyl]indolin-5-carboxylic acid (303 mg) and the mixture was stirred at room temperature overnight (Solution A). On the other hand, in anhydrous methanol (5 ml) was dissolved DL-2-amino-4-phosphonobutyric a ~~.~~~ J~~a~
acid at U °C and then thionyl chJ.ori.de (:L rnl.) was gradually added to the solution at. the sarne temperature. After the mixture was returned to room temperature and stirred overnight, the solvent was removed under reduced pressure.
The residue obtained was dissolved in anhydrous dimethylformamide (Solution B). Then, Solutions A and B
were mixed. To the mixed solution was further added triethylamine (505 mg) and the mixture was stirred at room temperature for 3 days. Then, the solvent was removed under reduced pressure to obtain a residue (700 mg). From the residue obtained, 100 mg thereof was taken and fractionation was carried out by using a high performance liquid chromatography (column: YMC-A323) with an eluent of water : acetonit rile : trifluoroacetic acid = 87.5 : 12.5 1 to obtain 'the title compound (2 mg).
1H-NMR (D20, b): 1.7-2.3 (4H, m), 3.06 (2H, t, J=8Hz), 3.58 (2Ii, 't, J=8Hz) , 3. 84 (3H, s) , 4 . 36 (1H, m) , 4 .70 (2H, s) , 6. 63 (1I-I, d, J=8. 6I3z) , 7 . 60 (2H, m) , 8 .78 (1H, s) .
Reference example 44 Synthesis of methyl 4-amino-~ hxdroxy-benzoate In methanol (40 ml) was suspended 4-amino-3-hydroxy-benzoic acid and to the suspension was passed through a hydrogen chloride gas for 10 minutes, and the mixture was stirred at room temperature for 6 hours. Deposited pre-cipitates were collected by filtration, washed three tiine5 with ether and then vacuum dried to obtain the title compound (4.15 g) .
1H-NMR (CDC13: b) : 3. 83 (3H, s) , 6. 68 (1H, d, J=8Hz) , 7.41 (2H, m) .
Reference example 45.
Synthesis 4-methoxycarbonyl-2-hydroxy-chloroacetyl-anilide In dichloromethane (30 ml) were dissolved. the compound (1.0 g) of Reference example 44 and triethylamine (4.0 ml) and the solution was made 0 °C. To the solution was added ~dJj~J J~~~.~~~
dropwise a dichloromethane so:l.ution (5 ml) of chloroacetyl chloride (1.0 ml) under nitrogen atmosphere over 8 m9.nutes, and the mixture was stirred at 'the same temperature for one hour. Then, to the reaction mixture was added a saturated aqueous ammonium chloride solution and deposited precipi-tates were collected by filtration. Recrystallization was carried out by using chloroform-acetone to obtain the title compound (0.83 g).
1H-NMR (CDC13: 8) : 3. 90 (3H, s) , 4 .25 (2II, s) , 7.52 (2H, m) , 8.37 (1I-I, d, J=8Hz) .
Reference Qxample 46 ~,~rnthesis of methyl 3-oxo-3~4-di.h~~~ 2I-I-1~4~1,~.n_z~
xazin-7-carbQxyl~
Tn ethanol (300 ml) was suspended the compound (6.0 g) of Reference example 45, and to 'the suspension was added potassium acetate (5.0 g) and -the mixture was refluxed under nitrogen atmosphere for 90 minutes. After cooling the reaction mixture, precipitates were collected by filtration and vacuum dried to obtain the title compound (5.2 g) .
1H-NMR (DMSO-d6: b): 3.80 (3H, s), 4.63 (2H, s), 6.95 (1H, m), 7.43 (1H, m), 7.56 (1H, m).
Reference examgle 47 Synthesis of methyl 3,4-dihydro-2H-1,4-benzoxazin-7-carboxylate Under nitrogen atmosphere, to tetrahydrofuran (30 ml) were added tetrahydrofuran~borane complex (1 [M] solution, 10 ml) and the compound (440 mg) of Reference example 46 at 0 °C, and the mixture was stirred at room temperature for 15 minutes, and then refluxed for 4 hours. The reaction mixture was cooled to room temperature and 6N-hydrochloric acid (2.7 ml) was added thereto. The reaction mixture was condensed under reduced pressure and poured into water and made alkaline with 2N-sodium hydroxide aqueous solution.
Then, the title compound was extracted with ethyl acetate and the organic layer obtained was washed with a saline ~;a~C~~u solution and dried over magnesium sulfate. After removing the solvent under reduced pressure, the residue obtained was subjected to silica gel column chromatography. The residue was developed and eluted with a mixed solvent of hexane : ethyl acetate = 3 : 2 to obtain the title compound (310 mg) .
1H-NMR (CDC13: 8) : 3. 46 (2H, m) , 3.84 (3H, s) , 4 .22 (2H, t, J=4 .4Hz) , 4 .30 (:LH, m) , 6.53 (1H, d, J=
9.8 Hz), 7.45 (1I-I, s), 7.47 (1H, d, J=
9 . 8I-I z ) .
Reference example 98 ~ynthesi s of methyl N-carbobenzoxv-3, 4-~dihydro-2f-I-1, 4-.
benzgxaz in-7-carb~x~lat~
In tetrahydrofuran (50 ml) was dissolved the compound (1.4 g) of Reference example 47, and to tree salution was added gradually sodium hydride (700 mg) and the mixture was stirred at room temperature for 30 minutes. Then, carbo-benzoxy chloride (3 ml) was added to the mixture and t he mixture was stirred overnight. After several drops of water were added to the reaction mixture, the reaction mixture was poured into cool water and the title compound was extracted with ethyl acetate. The organic layer obtained was dried over sodium sulfate and the solvent was removed under reduced pressure. The residue was recrystallized from hexan-ethylacetate to obtain the title compound (1.79 g).
1H-NMR (CDC13: 8) : 3. 85 (3H, s) , 3. 91 (2H, m) , 4 .25 (2H, m) , 5. 34 (2H, s) , 7 . 35 (5H, m) , 7 .53 (2H, m) , 7 . 99 (1H, m) .
Reference exdmple 99 synthesis of N-carbobenzoxy-3 9-dihydro-2H-1,,4-bgnzo-xazin-7-carboxxlic acid In ethanol (50 ml) was suspended 'the compound (:L .79 g) of Reference example 98, and to the suspension was added 1N-sodium hydroxide aqueous solution (8.2 ml) and the mix-ture was stirred overnight. The solvent was removed under 1.~ ~ ~~ ~P~
reduced pressure and the residue obtained was dissolved in water (20 ml). Then, 1N-hydrochloric acid was gradually added thereto to adjust pI-I=2, and deposited precipitates were collected by filtration and vacuum dried to obtain the title compound (1.39 g).
1H-NMR (CDC13: 8) : 3. 82 (2H, m) , 4 . 14 (2H, m) , 5 . 13 (2H, s) , 7.2-7 .7 (7H, m) , 7. 95 (1H, m) .
Reference Axam~le 50 Synthesis of dime hvl N-- (3, 4-dihydro-2H-1, ~I-benzo-xazin-7-Carbonyl)-L-a-amino-ac~ipatg In thionyl chloride (5 m1) was suspended t he compound (800 mg) of Reference example 49, and a catalytic amount of dimethylformamide was added t o the mixture and the mixture was stirred at room temperature for 2 hours. Then, excess thionyl Chloride was removed under reduced pressure, and the residue was triturated with hexane. After Collecting the resulting crystals by filtration, the Crystals were dissolved in dichlorometha.ne (20 ml) and to the dichloro-methane solution was added dropwise an aqueous solution containing L-a-amino-adipic acid dimethyl ester hydrochlor-ide (1.0 g) and triethylamine (1.0 g) under ice-water cool-ing. The mixture was stirred at room temperature over-night, and the solvent was removed under reduced pressure.
To the residue was added a mixed solution of ethyl acetate and dil. hydrochloric acid under ice-water cooling. After stirring for 5 minutes, the organic layer was separated and then the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution. After drying over magnesium sulfate, the solvent was removed under reduced pressure. The residue obtained was dissolved in ethanol (80 ml) and after adding 10 o-palladium-carbon (900 mg), the mixture was stirred under hydrogen atmosphere at room temperature overnight. Palladium-carbon was removed by filtration using Celite, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel Column Chromatography by using chloroform - 61 _ a ! ~ :~ s , ~.) ~~3W,o~_ methanol = 100 . 1 as an eluent to obtain the title compound (230 mc!) .
1H-NMR (CDC13: 8) : 1 . 6-2. 1 (4I-l, m) , 2 .38 (2H, t, J=6. 8Hz) , 3.96 (2H, m) , 3. 68 (3H, s) , 3.7 7 (3H, s), 4.24 (2H, m), 4.72 (1H, m), 6.59 (1H, d, J=8.3Hz) , 7.33 (2H, m) .
Example 26 ,~ynthesi.s of dimet~l N- f 1- f (2 4=diamino) -6-pt ri. in-yl) meth~ll -3,, 4-dihydro-2H-1,L4-b nz ~xazin-7-~rbonyl.l -L-a.-amino-adipa~
In dimethylacetamide (3 ml) were suspended the corn-pound (200 mg) of Reference example 50 and 6-bromomethyl-2,4-di.aminopteridine hydrobromide~isopropanol adduct (226 mg), and the suspension was stirred at 60 °C for 6 hours.
After cooling, the reaction mixture was poured into a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel column chromatography by using a mixed solvent of chloroform : methanol = 10 . 1 as an eluent to obtain the title compound (260 mg).
1H-NMR (CD30D, b): 1.6-2.0 (4H, m), 2.36 (2H, t, J=6.8Hz), 3.58 (2H, m), 3.66 (3H, s), 3.76 (3H, s), 4.39 (2H, m), 4.67 (2H, bs), 9.73 (1H, m), 6.66 (1H, d, J=8.3Hz), 6.99 (1H, t, J=7.3Hz) , 7 .29 (2H, m) , 8 .70 (1H, s) .
Example 27 Synthesis of N-f1-f(2,4-diamino)-6-pteridinyl)methyll-3L4-dihvdro-2.H-1, 4-l~enzoxazin-7-carbonvl~ -L-a-amino-adipic acid The compound (260 mg) of Example 26 was dissolved in ethanol (12 ml), and 1N-sodium hydroxide aqueous solution (0.45 ml) was added to the solution at 35 °C and the mix ture was stirred at the same temperature for 4 hours.

~ , y~' / r ~J
After continua.ng stirring at 25 "C for 20 haurs, oaater (0.5 ml) was added to the reaction mixture and ethanol was removed under reduced pressure. The residue obtained was dissalved in water (6 rnl), adjusted to pH=3.7 with 1N-hydrochloric acid under ice-water cooling and allowed to stand overnight at cool place. Depasited precipitates were collected by filtration to obtain the title compound (176 mg ) .
1H-NMR (DMSO-d6, S) : 1 . 5-2 . 0 (4I3, m) , 2 . 14 (2H, t, J=
6.8Hz), 3.68 (2H, m), 4.28 (3H, m), 4.72 (2F-I, bs), 6.80 (1H, t, J=8.3Hz), 7.31 (2H, m), 8.13 (1H, d, J=7.3Hz), 8 . 71 ( 1I-I, s ) .
Reference~arnple ~1 Synthesis of a-methy-1 'y-l,~~nzy1 N~buyoxycarbonyl-'L-alutamate _Cn a dimethylformamide solution (75 ml) of y-benzyl N-t-butoxycarbonyl-L-glut amate (5.0 g) was suspended sodium hydrogen carbonate (2.5 g), and a dimethylformamide solu-tion (75 ml) of methyl iodide (10.5 g) was added to the suspension and the mixture was stirred at room temperature for 24 hours. After the reaction mixture was condensed under reduced pressure, the residue was poured into water (70 ml) and extracted with a mixed solution of ethyl acetate : n-hexane = 1 . 1. The organic layer was washed with water and dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel column chromatography by using ethyl acetate : n-hexane = 1 . 2 as an eluent to obtain the title compound (5.2 g).
1H-NMR (CDC13, &) : 1 .43 (9H, s) , 1 .92-2.01 (1H, m) , 2.11-2 . 37 (1H, m) , 2 . 42-2 .51 (2H, rn) , 3 . 73 (3H, s), 4.23-4.40 (1H, m), 5.12 (2H, s) , 7.35 (5H, s) .
Reference example 52 Synthesis of a-methyl N-t-butoxycarbonyl-L-gvlutamate-J
After adding 10 'a palladium-carbon (1..1 g) to a methanol solution (30 ml) of the compound (5.2 g) of Refer-ence example 51, the mixture was stirred under hydrogen atmosphere at room temperature for 15 hours. Palladiurn-carbon was removed by filtration using Celite and the solvent was removed under. reduced pressure. The residue obtained was dissol~red in a saturated aqueous sodium hydrogen carbonate solution and 'the solution was washed with chloroform. After separating the aqueous layer, it ZO was adjusted to pH=4 with 5o citric, acid and extracted with chloroform. The chloroform layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the title compound (3.9 g).
1H-NMR (CDC13, b) : 1 .94 (9H, s) , 1 . 89-2 . 04 (1H, m) , 2.09-2.27 (1H, m), 2.33-2.59 (2H, m), 3.75 (3I-I, s) , 4 .24-4 .44 (1H, m) , 5. 1 7-5.21 (1H, m) , 9.38 (1H, bs) .
Reference example 53 synthesis of methyl 4-amino-n-butyrate hydrochloride Into a methanol solution (20 ml) of 4-amino-n-butyric acid (1.0 g) was passed through a hydrogen chloride gas for 10 minutes and the mixture was stirred at room temperature for 5 hours. The solvent was removed under reduced pres-sure to obtain the title compound (1.5 g).
zH-DIMR (DMSO-d6, 8) : 1 . 83-2 .24 (2H, m) , 2 . 32-2 . 60 (2H, m) , 2..76-3.26 (2H, m), 3.61 (3H, s), 8.26 (2I-I, bs) .
Reference example 54 Synthesis of a-methyl N-t-butoxycarbQnvl-N~-l3-methoxycarbonylpropyl)-L-alutaminate To a tetrahydrofuran solution (5 ml) of the compound (518 mg) of Reference example 52 and triethylamine (0.33 ml) was added a tetrahydrofuran solution (1 m1) of isobutyl chlorocarbonate (0.31 m1) at -20 °C under nitrogen atmos-phere, and the mixture was stirred for 30 minutes. Then, after adding a tetrahydrofuran suspension (5 ml) of the '~~~t~''~~J~~~
compound (366 mg) of. Reference example 53 and triethylamine (0.33 ml) to the mixture, the mixture was stirred for one hour. The mixture was gradually returned to room tempera-ture and further stirred for 24 hours. The solvent was removed under reduced pressure and the residue obtained was dissolved in ethyl acetate. The ethyl acetate layer was washed successively with a 5 % citric acid aqueous solu-tion, a saturated aqueous sodium hydrogen carbonate solu-tion arid water, and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue obtained was subjected to silica gel column chromatography by using chloroform : methanol = 99 : 1 as an eluent t o obtain 'the title compound (575 mg).
1H-NMR (CDC13, 8) : 1 . 44 (9H, s) , 1 .78-1 . 99 (3H, m) , 2 . 16-2 .19 ( 1I-I, m) , 2 .22-2 .31 (2H, m) , 2 . 39 (2H, t, J=7.lHz), 2.70 (2H, q, J=6.5Hz), 3. 68 (3I-I, s) , 3.74 (3H, s) , 4 . 14-4.31 (1H, m), 5.35 (1H, bs), 6,43 (1H, bs).
Reference example 55 Synthesis of a-methyl N'-f3-methoxycarbonylpropyl)-L-glutaminate trifluoroacetate The compound (726 mg) of Reference example 54 was dissolved in trifluoroacetir_ acid (2 ml) under ice-cooling and the mixture was stirred for one hour. The solvent was removed under reduced pressure to obtain the title compound (754 mg) .
~H-NMR (CDC13 : CD30D = 20 . 1, 8) : 0.92 (2H, quint, J=
6.3Hz), 2.04-2.42 (4H, m), 2.50 (2H, t, J=6.3Hz), 3.17-3.24 (2H, m), 3.67 (3H, s) , 3. 81 (3I3, s) , 4 . 09-4 . 15 (1H, m) , 7.44 (1H, t, J=6.OHz).
Reference example 56 ~nthesis of a-methyl N-(1-carbobenzoxyi.ndolin-5-carY~onyl) -rr~- (3-methoxy-carbon~propyl) -L-g~lutaminate By adding thionyl chloride (5 ml) to 1-carbobenzoxy-indolin-5-carboxylic acid (599 mg) and the mixture was 1~~~ a~i~~t~
stirred at room temperature for 2 hours. Then, the mixture was evaporated to dryness under reduced pressure. The solid material obtained was dissolved in methylene chloride (7 ml), and to the solution was added an aqueous solution (3 ml) of the compound (754 mg) of Reference exarnple 55 and sodium hydrogen carbonate (534 mg) at room temperature and the mixture was stirred for 15 hours. Sodium hydrogen carbonate was added to the mixture until pH became 8 and insolubles were removed by filtration using Celite, and the methylene chloride layer was collected by separation. 'The methylene chloride layer was washed successively with 1N-hydrochloric acid and water, and dried over sodium sulfate.
The solvent was removed under reduced pressure and the residue obtained was subjected to silica gel column a.5 chromatography using chloroform : methanol = 99 : 1 as an eluent to obtain 'the title compound (474 mg).
1H-NMR (CDC13, 8) : 1 .81 (2I-I, quint, J=7.OI-Iz) , 2.09-2.27 (4F3, m) , 2 .34 (2I-I, t, J=7. 3Hz) , 3 . 15 (2H, t, J=9.OHz), 3.27 (2H, t, J=5.4Hz), 3. 65 (3H, s) , 3.77 (3H, s) , 4 .10 (2F-I, t, J=8 . 8Hz) , 4 . 65-4 .75 (1I-I, m) , 5.28 (2H, s), 6.38-6.45 (1H, m), 7.36-7.46 (6H, m), 7.66-7.68 (2H, m).
Reference example 57 Synthesis of a-methyl N-(indolin-5-carbonvll-N'-(3-methoxycarbonylpropyl)-L--alutaminate After adding 10 ~ palladium-carbon (90 mg) to a methanol solution (10 ml) of the compound (470 mg) of Reference example 56, the mixture was stirred under hydrogen atmosphere at room temperature for 15 hours.
After removing palladium-carbon by filtration using Celite, the solvent was removed under reduced pressure to obtain the title compound (350 mg).
1H-NMR (CDC13, 8): 1.66-2.48 (8H, m), 2.81-3.30 (4H, m), 3.50 (2H, t, J=6.OHz), 3.64 (3H, s), 3.73 (3I-I, s) , 4 .49-4 .81 (1H, m) , 6.51 g , x ~1,~l5i ~~
(2I-I, d, J =9. OI-Iz) , 6 . 62-6 . 84 (lfl, m) , '7 . 21-7 . 33 (1H, rn) , 7 . 42--'7 , 57 (2H, rn) .
sample 28 ~vnthes~ c~.~~1?yl N- f 1- f (2 ~ 4-~ir~mino-6- ri ins yl)methvll-inc~olin-5-carbon,~l,.LsL._-_~.~Y~arbonylpropvl~
)-L-alutamin~
In dimethylacetamide (7 ml) were suspended the com-pound (350 mg) of Reference example 57 and 6-bromomethyl-2,4-diaminopteridine hydrobromide~isopropanol adduct (414 mg), and the suspension was stirred at room temperature fox 24 hours. To the mixture was added triethylamine (0.29 rnl,) and the mixture was stirred for 10 minutes, it was subjected to silica gel column chromatography by using, as eluents, ethyl acetate~and then chloroform : methanol = 9 1 as an eluent to obtain the title compound (263 mg).
lI-I-NMR (DMSO-ds : CDC13 = 7 . 3, 8) : 1.67 (2H, quint, J=
6.9Hz), 1.91-2.14 (2H, m), 2.19-2.33 (4H, m), 2.97-3.13 (4H, m), 3.56-3.59 (5H, m), 3.65 (3H, s), 4.34-4.49 (1H, m) , 4 .55 (2H, s) , 6 . 67 (1I-I, d, J=8.3Hz) , 7.32 (4H, bs), 7.62-7.66 (2H, m), 7.84 (1H, t, J=8.3Hz), 8.33 (1H, d, J=7.3Hz), 8.71 (1H, s) .
Example 29 synthesis of N-f1-fl2,4-diamino-6 ~t'eridinyl)-methyll-indolin-5-carbonyll-N'-(~-carboxypropvll-L-alutamin~
To a methanol (5 ml) solution of the compound (250 m~c) of Example 28 was added 1N-sodium hydroxide aqueous solu-tion (0.95 ml) and the mixture was stirred at room tempera-ture for 2.0 hours. While maintaining the temperature of water bath to 30 °C or lower, the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel colurnn chromatography using chloroform : methan-ol . 28 o aqueous ammonia =- 5 . 9 . 1 as an eluent to obtain brownish solid material. The solid material obtain-ed was dissolved in water and insolubles were filtered off, _ 67 _ 6 k! :!
1~~.3~ia~J~~~
and t he solution was adjusted to pH=3.7 with 1N-hydrochlor-ic acid. Deposited brownish precipitates were cal7.ected by filtration to obtain the title compound (199 mg).
1H--NMR (DMSO-d~ : CDC13 = 9 . 1, 8) : 1 .61 (2H, quint, J=
7.OFIz), 1.89-2.08 (2H, m), 2.13-2,30 (4H, m) , 2 . 95-3. 10 (4H, m) , 3.58 (2H, t, J=8.OHz), 4.25-9.38 (1H, m), 9.55 (2H, s), 6.70 (1H, d, J=8.3Hz), 7.61-7,65 (2H, m) , 7 . 85 (1~I, t, J=5. 8Hz) , 8 .21 (1H, d, J=7 .3Hz) , 8.73 (1I-I, s) .
Example 30 SSynthesis of ammonium N-f1-f(2,~I=diamino-6-gteridin-yl ) methyl l -indolin-5~carb_onyl l -hornc~~s eate To a benzene suspension (6 ml) of homocysteic acid hydrobromide ( 330 ml ) were added triethylamine ( 8 70 ~.7. ) and chlorot.rimethyl silane (630 ~.1) under nitrogen atmosphere, and 'the mixture was stirred at room temperature for 3 days.
Deposited precipitates were filtered off and the filtrate was condensed to obtain silylated homocysteic acid (950 2p mg). Under a nitrogen atmosphere, in dimethylformamide (18 ml) were dissolved dimethyl cyanophosphonate (213 ~L1) and triethylamine (172 ~Ll) , and 1- [ (2, 9-diamino) -6-pteridinyl) -methyl]indolin-5-carboxylic acid (170 mg) was added little by little at room temperature and the mixture was stirred at the same temperature for 3 hours. To the solution was added a dimethylformamide solution (2 ml) of silylated homocysteic acid (950 mg) and the mixture was stirred at room temperature for 2 days. Then, water (1 ml) was added to the reaction mixture and the solvent was removed under reduced pressure. To the residue was added 3 % ammonium bicarbonate aqueous solution and after removing insolubles by filtration, the filtrate was applied to DEAF-cellulose column, washed with water and eluted by 3 % ammonium bicarbonate aqueous solution to obtain the title compound (18 mg) .

1H-NMR (D2U, b) : 2 .0-2..4 (2fi, m) , 3.15 (4H, m) , 3.49 (2fI, m) , 4 .34 (1H, m) , 4 . 49 (2H, s) , 6 . 66 (1FI, m) , 7 .55 (2.H, m) , 8 . 69 (1I-I, s) .
Reference exampl~~$
SynthPSis of methyl 3~ ~l-dih~ro-2H-1, 4-benzQthiazi.ne-~rboxvlate A mixture of 2-aminobenzothiazol-6_carboxylic acid (15 g), potassium hydroxide (22 g ) and water (22 g) was re-fluxed under a nitrogen atmosphere :Eor 3 hours. The mixture was cooled to room temperature, and water (20 ml), 1,2-dibromoethane (40 ml) and hexadecyltributylphosphoniurn bromide (3 g) were added thereto and the mixture was refluxed for 6 hours. The mixture was cooled t o room temperature, water and. chloroform were added theret a and insolubles were removed by decantation. The aqueous layer was adjusted to pH=3 with hydrochloric. arid and the chloroform layer was collected by separation. The chloroform layer obtained was washed with a saturated saline solution and dried over anhydrous sodium sulfate.
The solvent was removed under reduced pressure and the residue was dissolved i.n methanol. To the solution was passed through a hydrogen chloride gas for 10 minutes and the mixture was stirred at room temperature overnight. To the residue obtained by removing the solvent under reduced pressure were added water and ethyl acetate, and the aqueous layer was adjusted to pH=5 with 1N sodium hydroxide aqueous solution and then extracted with ethyl acetate.
The organic layer obtained was washed with a saturated saline solution and dried over anhydrous sodium sulfate.
The solvent was removed under reduced pressure and the residue was subjected t o silica gel column chromatography by using ethyl acetate : hexane = 1 . 4 as an eluent to obtain the title compound (721 mg).
1H-NMR (CDC13, b): 2.9-3.1 (2H, m), 3.6-3.9 (2H, m), 3.83 (3H, s) , 4 . 0-5. 0 (1H, br) , 6.39 (1H, d, J=8.4Hz), 7.45-7.75 (2H, m).
- 69 _ Refer~n~~xample 59 Synthesis df N-carbobenzoxy-3,4-dihydrg-2H-1,4-benzo-thiazine-7-carboxylic acid The compound (313 mg) of Reference example 58 was dis-solved in tetrahydrofuran (10 ml) and to the solution was gradually added sodium hydride (180 mg), and the mixture was stirred at room temperature for 20 rninutes. Then, carbobenzoxy chloride (1.3 ml) was added t:o the mixture and the mixture was stirred overnight. After adding water to the mixture, the mixture was extracted with ethyl acetate.
The organic layer obtained was dried over anhydrous sodiurn sulfate and then the solvent was removed under reduced pressure. The residue was applied to silica gel column chromatography by using ethyl acetate : hexane = 1 . 10 as an eluent, and methyl N-carbobenzoxy-3,9-dihydro-2H-1,4-benzothiazine-7-carboxylate obtained was suspended in ethanol (15 ml). To the suspension was added 1N-sodium hydroxide aqueous solution (1.9 ml) and the mixture was stirred overnight. The solvent was removed under reduced pressure, and the residue obtained was dissolved in water (20 m1). Then, 1N-hydrochloric acid was gradually added to adjust pF-I=3 and the mixture was extracted with chloroform.
The organic layer was washed with a saturated saline solu-tion and dried over anhydrous sodium sulfate. The residue obtained by removing the solvent under reduced pressure was applied to silica gel column chromatography by using chloroform : methanol = 95 : 5 as an eluent to obtain the title compound (208 mg) .
1H-NMR (CDC13 : CD30D = 9 : 1, 8): 3.0-3.3 (2H, m), 3.8-9.1 (2H, m), 5.25 (2H, s), 7.3-7.9 (8H, m).
Reference example 60 ~,ynthesis of diethyl N- (3~4-dihydro-2H-1~ 4-benzo-thiazine-7-carbonyl)-L-alutamat~
To a dimethylformamide solution (5 ml) containing the compound (208 mg) of Reference example 59, diethyl L-glutamate hydrochloride (151 mg), 1-hydroxybenzotriazole (90 mg) and N-rnethyl.morpholine (70 ~1) was added dicyclo-hexylcarbodii.mide (195 mg) under ice-cooling, and the mixture was stirred at the sarne temperature for one hour and then at room temperature for overnight. To tine mixture was added ethyl acetate, and precipitates were filtered off and the filtrate was washed successively with an aqueous sodium hydrogen carbonate solution and a saturated saline solution and dried over anhydrous sodium sulfate. The residue obtained by removing the solvent under reduced pressure was applied to silica gel column chromatography by using ethyl acetate : hexane = 1 . 2 as an eluent, and diethyl N-[N'-carbobenzoxy-3,4-di.hydro-2I-I-1,9-benzothia-zine-7-carbonyl]-L-glutamate obtained was dissolved in ethanol (20 m1). After adding 10 % palladium-carbon (700 mg) to the solution, the solution was stirred under hydro-gen atmosphere at room temperature overnight. Palladium-carbon was removed by filtration using Cel.ite, and the solvent was removed under reduced pressure. The residue obtained was subjected to silica gel column chromatography by using a mixed solvent of ethyl acetate : hexane = 2: 3 as an eluent to obtain the title compound (83 mg).
1H-NMR (CDC13 : CD30D = 9 : 1, 8): 1.25 (6H, m), 2.0-2.7 (4H, m), 2.9-3.1 (2H, m), 3.6-3.8 (2H, m) , 3. 9-4 .4 (5H, m) , 6.45 (1I-I, d, J=
8.OHz), 7.2-7.6 (2H, m).
Example 31 Synthesis of diethyl N-(1-x((2,4-diamino)-6-pteridin-yl)methyll-3,4-dihydro-2H-1,4-benzothiazine-7-carbonyll-L-alutamat~
T.n dimethylacetamide (3 ml) were suspended the com-pound (83 mg) of Reference example 60 and 6-bromomethyl-2,9-diaminopteridine hydrobromide~isopropanol adduct (85 mg), and the suspension was stirred at 60 °C for 3 hours and then at 100 °C for 30 minutes. After cooling, 'the reaction mixture was poured into a saturated aqueous sodium hydrogen carbonate solution and extracted three times with ~~~S~7c~~
chloroform. The organic .layer way; dri.ed over anhydrous sodium sulfate a.nd the solvent was .r.emoved under reduced pressure. The residue obtained was applied 'to silica gel column chromatography by using a mixed solvent of chloro-form : methanol = 93 . 7 as an eluent to obtain the title compound (21 mg) .
1H-NMR (CDC13 : CD30D = 9 . 1., b) : 1 .26 (6I-I, rn) , 2 .0-2 .3 (2H, rn) , 2 . 46 (2H, m) , 3. 12 (2H, m) , 3. 91 (2H, m) , 4.1-4 .3 (4H, m) , 4 . 62 (lI-I, m) , 4 .78 (2I-I, s) , 6 . 69 (1H, d, J=8. 9f-Iz) , 7.04 (lH,m), 7.60 (1H, d, J=2.2I-Iz), 8.66 (1H, s) .
Example ,~,.2 Synthesis of N-f1-f((2,4-di~mino)~6-pte.ridinyl.~
m~L3 ~ 4-dihydrQ-2H-1-~4-benz ~iazinE-7-c~rbonyl~L-alutamic acid The compound (20 mg) of Example 37. was dissolved in ethanol (2 ml), and 1N-sodium hydroxide aqueous solution (170 ~.1) was added to the solution at 35 °C and the mixture was stirred at the same temperature for 4 hours. After continuing stirring at 25 °C for 20 hours, water (0.5 ml) was added to 'the reaction mixture and ethanol was removed under reduced pressure. The residue obtained was dissolved in water (6 ml), adjusted to pH=3.7 with 1N-hydrochloric acid under ice-water cooling and allowed to stand overnight at cool place. Deposited precipitates were collected by filtration to obtain the title compound (18 mg).
1H-NMR (DMSO-d6, b): 1.8-2.2 (2H, m), 2.30 (2H, m), 3.18 (2I-I, m) , 3 . 95 (2H, m) , 4 .37 (1H, m) , 4 .76 (2H, s) , 6.79 (1H, d, J=8 . 8Hz) , 7.42 (1H, m), 7.59 (1H, d, J=2.OHz), 8.22 (1H, d, J=7.3Hz) , 8 .67 (1H, s) .
Example 33 synthesis of N-fl-f(2,4-diamino-6 ~teridinyl)methyll--1-oxo-3 ~4-dihydro-2H-1 4-benzothiazine-7--carbonyl lL-~lutamic acid The compound (:LO md) of Example 32 was suspended in water (1 ml) and :LN-~sodi.urn hydroxide aqueous solution was added thereto to dissolve the ma.xture. Under ice-cooling, 0.5M sodium metaperi.odate (50 ~.1) was added to the solution and the mixture was stirred at -the same temperature for 5 hours. The reaction mixture was adjusted to pH=3.5 and deposited precipitates were collected by filtration to obtain the title compound (4 mg).
1H-NMR (DMSO-d6, 8) : 1 . 8-2 . 2 (2H, m) , 2 . 33 (2EI, m) , 2 . 9-3. 3 (2H, m) , 3 . 8-4 .0 (1I-I, m) , 4 .2-4 .5 (2H, m) , 4.83 (1H, d, J=17. 1F-Iz) , 5.19 (1H, d, J=17.1Hz) , '7.07 (1I-I, d, J=9.:3Hz) , 7.7-7.9 (1H, m), 8.1-8.2 (1H, m), 8.42 (1H, m) , 8.72 (1H, s) .
IR (KEir) vmax 2800-3600, 1644, 1608, 1552, 1504 and 1008 cm-1 FAB M S 515 ( M-H 1 ) '~
Reference example 61 Synthesis of benzyl N-t-bu~oxycarbonyl-N~-methane ~ulfonvlc~lutaminate In tetrahydrofuran (268 ml) were dissolved N,N'-carbonyldiimidazole (13.6 g) and a-benzyl [N-(t-butoxy-carbonyl)glutamate] (25 g) and the mixture was stirred under ice-cooling for one hour. Then, the solution was added dropwise to a tetrahydrofuran (132 ml) solution containing methanesulfoneamide (20.5 g) and 1,8-diazabi-cyclo[5.4.0]-7-undecene (32.9 g) under ice-cooling. After dropwise addition, the mixture was returned to room tem-perature and stirred for 4 days. After adding 500 ml of 1N
hydrochloric acid to the mixture, it was extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and the solvent was removed under reduced -pressure to obtain the title compound (30 mg).
1H-NMR (CDC13, 8) : 1 .42 (9H, s) , 1 . 8-2. 8 (4H, m) , 3.25 (3H, s), 4.32 (1H, m), 5.14 (2H, s), 7.40 (5H, s) .
- 73 _ ~1~~~~6~.
~~rence x m ~L_e 62.
S_ynth~,~ix~F benzv_,l N!-mQt~han~~~lf~rylg,l.~~~~n_ate~tri-flyro~:etate The compound (4.65 g) of Reference example 61 was dissolved in t rifl.uoroacetic acid (60 ml) and the solution was stirred at room 'temperature for one hour. Then, the reaction mixture was condensed at 30 °C and triturated by adding ether to obtain the title compound (4.5 g).
1H-NMR (CDC13, 8) : 1 . 8-2 . 8 (4I-I, m) , 3.20 (3H, s) , 4 .50 (1H, m) , 5.28 (2H, s) , 7 . 42 (5H, s) .
Example 34 Synthesis of benzyl N-(1-f(2,4-diamino-~6-p ri. in-yl)methyll i.n~Qlin-5-carbOnVl.1_ N'-methan_~s"~l~n~l.yt~Lni.n~~~, Tn dimethylformamide (18 ml) were dissolved diethyl cyanophosphonate (2a.3 ~1) and triethyl.amine (172 ~.1) under nitrogen atmosphere, and to the sol~.ztion was added 1-((2,4-diamino--6-pt eridinyl)methyl]indolin-5-carboxylic acid (160 mg) little by little and the mixture was stirred at the same temperature for 3 hours. To the mixture was added a dimetylformamide solution (3 ml) containing the compound (450 mg) of Reference example 62 and triethylamine (180 )11) and the mixture was stirred at room temperature for 3 days.
Then, water (1 ml) was added to the reaction mixture and the solvent was removed under reduced pressure. The residue was subjected to silica gel column chromatography by using a mixed solvent of chloroform : methanol . aqueous ammonia = 15 : 5 . 1 as an eluent to obtain the title compound (60 mg).
1H-NMR (DMSa-d6, 8) : 1 . 9-2 . 1 (2H, m) , 2 . 3-2 .5 (2H, m) , 2 . 98 (2H, t, J=8.6I-iz) , 3.28 (3H, s) , 3.55 (2H, t, J=8.6Hz), 4.21 (1H, m), 9.53 (2H, s) , 5. 04 (2H, s) , 6.71 (1H, d, J=8.6Hz), 7.34 (5H, s), 7.55 (2H, m), 8.72 (1H, s) .
Example 35 ~4~~S~~i~a Synthesi.s~~f. N- f 1- L 2~n-d.i~mi.no-~pter.:idinyl) methyll indolin-5-carbon,~l ~~N' =m~t,-h~nesulfQyy'lc~lutamine The compound (25 mg) of Example 34 was suspended in ethanol (5 ml), and 1N-sodium hydroxide aqueous solution (200 ~l) was added to the suspension and the mixture was stirred at room temperature overnight. Then, water (O.S
ml) was added to the mixture and ethanol was removed under reduced pressure. 'rhe residue obtained was dissolved in water (6 ml) and the solution was adjusted to pI-1=3.7 with 1N-hydrochloric acid under ice-water cooling and allowed to stand at cold place overnight. Deposited precipitates were collected by filtration to obtain the title compound (20 mg) .
1H-NMR (DMSO-dg, 8) : 1 , 9-2 . 1 (2I-I, m) , 2 .2-2 . 9 (2H, rn) , 3 . 00 (2H, t, J=8.6Hz), 3.19 (3H, s), 3.60 (2H, t, J=8.6Hz), 9.35 (1H, m), 4.56 (2I3, s) , 6.70 (1H, d, J=8.6I-Iz) , 7.63 (2H, m), 8.75 (1H, s).

Claims (10)

The embodiments of the invention, in which an exclusive property or privilege is claimed are defined as follows:

1. A compound represented by the formula:
wherein R1 represents CH2, CH2CH2, CH2O, CH2S or CH2SO; R2 represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms or a benzyl group; n represents an integer of 1 to 4; R3 represents a group of formula PO3H2, SO3H, COOR4 in which R4 represents hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, a group of formula NHCOR5 in which R5 represents an unsubstituted or substituted phenyl group, or a group of formula CONR6R7 in which R6 represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, and R7 represents a lower alkyl group having 1 to carbon atoms, an unsubstituted or substituted phenyl group, an unsubstituted or substituted carboxylalkyl group or a lower alkylsulfonyl group.

2. A compound according to claim 1, wherein R3 is PO3H2, SO3H or COOR4, R4 having the same meaning as defined in claim 1.

3. A compound according to claim 1, wherein R3 is NHCOR5, R5 having the same meaning as defined in claim 1.

4. A compound according to claim 1, wherein R3 is CONR6R7, R6 and R7 having the same meanings as defined in claim 1.

5. An antirheumatic pharmaceutical composition comprising as active ingredient a compound according to claim 1, together with a pharmaceutically acceptable carrier therefor.

6. A psoriasis curing pharmaceutical composition comprising as active ingredient a compound according to claim 1, together with a pharmaceutically acceptable carrier therefor.

7. A carcinostatic pharmaceutical composition comprising as active ingredient a compound according to claim 1, together with a pharmaceutically acceptable carrier therefor.

8. A process for preparing a compound represented by the formula (I) wherein R1, R2, R3 and n have the same meanings as defined in claim 1, which comprises reacting a compound represented by the formula (I) -1:
wherein R1 has the meaning as defined above and A1 represents a protective group, with a compound represented by the formula (I) - 2:

wherein R2, R3 and n have the meanings as defined above, to obtain a compound represented by the formula (I)-3:
wherein R1, R2, R3, n and A1 have the meanings as defined above, deprotecting the compound of formula (I)-3 and reacting it with a compound represented by the formula (I)-4:
wherein X represents a halogen atom.

9. A process for preparing a compound represented by the formula (I):
wherein R1, R2, R3 and n have the same meanings as defined in claim 1, which comprises reacting a compound represented by the formula (I) -4 wherein X represents a halogen atom, with a compound represented by the formula (I)-5:

wherein R1 has the meanings as defined above and R' represents hydrogen atom or a lower alkyl group, to obtain a compound represented by the formula (I)-6:

wherein R1 and R' have the meanings as defined above, and when R' is a lower alkyl group, hydrolyzing the compound of formula (I) - 6 and then reacting it with a compound represented by the formula (I)-2:

wherein R2, R3 and n have the meanings as defined above.

10. A process for preparing a compound represented by the formula (I'):

wherein R1, R2 and n have the same meanings as in claim 1 and R' represents a hydrogen atom or a lower alkyl group, which comprises reacting a compound represented by the formula (I)-7:

wherein R2 and n have the meanings as defined above and A2 represents a protective group, with a compound represented by the formula (I)-8:

to obtain a compound represented by the formula (I)-9:

wherein R2, n and A2 have the meanings as defined above, and after deprotecting the compound of formula (I) - 9, reacting it with a compound represented by the formula (I)-1:

wherein R1 has the meaning as defined above and A1 represents a protective group, to obtain a compound represented by the formula (I) - 10:

wherein R1, R2, n and A1 have the same meanings as defined above, reacting it with a compound represented by the formula (I) - 4:

wherein X represents a halogen atom, and subjecting the resulting compound to ring opening reaction.